A method for constructing a 3D-printed liver organoid scaffold loaded with fh1 alginate sodium microspheres assembled mesenchymal stem cells

Abstract

The application discloses a construction method of a 3D printing liver organoid support loaded with mesenchymal stem cells assembled by FH1 sodium alginate microspheres, and comprises the following steps: preparing CaO2@ZIF-8@SL; preparing FH1-loaded sodium alginate microspheres FH1@SA; and preparing a 3D printing liver organoid support. The 3D printing liver organoid support prepared by the method can significantly enhance the differentiation of human umbilical cord mesenchymal stem cells into functional stem cells, and can be used for constructing a human liver organoid model.

Description

Technical Field

[0001] This invention belongs to the field of liver organoid construction, specifically relating to a method for constructing a 3D-printed liver organoid scaffold loaded with FH1 sodium alginate microspheres and assembled with mesenchymal stem cells. Background Technology

[0002] The liver is a vital metabolic center in the human body, participating in various pathological and physiological processes. When the liver suffers acute injury, its powerful regenerative capacity can repair up to 70% of the damaged area. However, chronic diseases resulting from acute injury, such as viral infections and alcoholic fatty liver disease, can lead to irreversible loss of liver function, ultimately resulting in end-stage liver disease. For patients with end-stage liver disease, the most effective clinical treatment is liver transplantation. Unfortunately, the number of available liver donors is far less than the demand from patients, causing approximately 20% of patients to die while waiting for a donor. Furthermore, the waiting period for a suitable liver donor is a heavy psychological burden for patients, as they cannot predict whether they will receive a suitable liver at the right time, often leading to poor prognoses. Given the shortage of liver donors, there is an urgent need to find a new organ model for clinical treatment research.

[0003] For over a decade, researchers have focused on developing functional hepatocytes for regenerative medicine research in vitro. However, as organoids have increased in size, passive diffusion has proven insufficient to provide adequate nutrients and oxygen while simultaneously clearing metabolites, ultimately leading to tissue necrosis due to hypoxia. Three-dimensional bioprinting (3D) technology has opened new avenues for the programmable deposition of mammalian cells in bioactive hydrogels. This innovation facilitates the development of 3D biological structures that closely mimic the complexity and heterogeneity of natural tissues, providing a platform for the in vitro generation of tissues and organs. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by using 3D bioprinting technology to assemble mesenchymal stem cells and sodium alginate microspheres loaded with FH1 to construct a human liver organoid model. This model has the ability to sustainably release FH1, a drug that promotes hepatocyte function.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a method for constructing a 3D-printed liver organoid scaffold loaded with FH1 sodium alginate microspheres and assembled with mesenchymal stem cells, comprising the following steps:

[0007] S1. Preparation of CaO2@ZIF-8@SL;

[0008] S2. Preparation of sodium alginate microspheres FH1@SA loaded with FH1;

[0009] S3. Fabrication of 3D-printed liver organoid scaffolds;

[0010] In a preferred embodiment, step S1 includes: adding CaO2 and 2-methylimidazole to water, mixing with Zn(CH3COO)2 and sodium lignosulfonate, incubating overnight, harvesting the solid and washing with deionized water, and then drying at room temperature to obtain CaO2@ZIF-8@SL.

[0011] Preferably, in step S1, the weight ratio of CaO2, 2-methylimidazole, Zn(CH3COO)2, and sodium lignosulfonate is (30-35):(3000-3500):(75-85):(8-12), and the most preferred weight ratio is 32.3:3200:81:10.

[0012] In a preferred embodiment, step S2 includes:

[0013] S201, calcium chloride solution and ethylenediaminetetraacetic acid solution are mixed evenly, then the pH is adjusted to 7.2, and finally ultrapure water is added to make up the volume to obtain Ca-EDTA solution;

[0014] S202. Dissolve FH1 in DMSO to obtain FH1 solution, then mix it with sodium alginate aqueous solution to obtain FH1@SA solution.

[0015] S203. Add the FH1@SA solution to an equal volume of Ca-EDTA solution and mix thoroughly to obtain an FH1@SA / Ca-EDTA solution.

[0016] S204. Inject the FH1@SA / Ca-EDTA solution and 2% microdroplet-generating oil into a syringe respectively, and extrude them through a microfluidic chip to form microspheres. Collect the generated microdroplets and then let them stand to solidify, thereby forming hydrogel microspheres. Then, take out the bottom microdroplet-generating oil, add a demulsifier, centrifuge and take out the bottom demulsifier. Finally, wash the microspheres with PBS and collect the FH1-loaded sodium alginate microspheres FH1@SA.

[0017] More preferably, in step S201, 1.2 mL of 2M calcium chloride and 4.8 mL of 0.5M ethylenediaminetetraacetic acid solution are mixed evenly, then 2M sodium hydroxide solution is added to adjust the pH to about 7.2, and finally ultrapure water is added to make up to 10 mL to obtain Ca-EDTA solution.

[0018] More preferably, in step S202, 10 mg of sodium alginate and 1 mg of FH1 are dissolved in 0.5 mL of deionized water and mixed evenly, and placed at 60 °C until completely dissolved to obtain FH1@SA solution.

[0019] In a preferred embodiment, step S3 includes:

[0020] Methacrylamide gelatin and methacrylamide hyaluronic acid were dissolved in deionized water containing LAP and lemon yellow and mixed evenly. Then, CaO2@ZIF-8@SL and FH1@SA were added and mixed evenly to obtain a hydrogel solution. HUCMSC cells were then added to obtain bio-ink. The prepared bio-ink was used for printing and cleaning to obtain a 3D-printed liver organoid scaffold assembled with FH1 sodium alginate microspheres.

[0021] In a preferred embodiment, the methacrylamide gelatin is prepared by the following steps:

[0022] Methacrylic anhydride was added dropwise to PBS buffer containing gelatin and the mixture was magnetically stirred. After the reaction was complete, the mixture was dialyzed in deionized water to remove byproducts. The dialyzed reaction solution was collected, centrifuged to remove the precipitate, and the supernatant was collected and freeze-dried to obtain the final product, methacrylamide gelatin.

[0023] More preferably, the concentration of gelatin in PBS buffer is 0.1-0.5 g / mL, and most preferably 0.1 g / mL.

[0024] More preferably, the mass ratio of gelatin to methacrylic anhydride is (1-2):1, and most preferably 1.5:1.

[0025] Preferably, the methacrylamide hyaluronic acid is prepared by the following steps:

[0026] Hyaluronic acid was dissolved in deionized water and mechanically stirred until completely dissolved. Methacrylic anhydride was added, and the pH was adjusted to 8.5 with alkali. The mixture was stirred at room temperature, and then dialyzed and lyophilized to obtain methacrylated hyaluronic acid.

[0027] More preferably, the mass ratio of hyaluronic acid to methacrylic anhydride is 1:(1-3), and most preferably 1:2.6.

[0028] In a preferred embodiment, in step S2, the mass ratio of methacrylated gelatin, methacrylated hyaluronic acid, CaO2@ZIF-8@SL nanoparticles, and FH1@SA is (25-75):10:5:2, more preferably 75:10:5:2.

[0029] As a preferred embodiment, the amount of HUCMSC cells added is 1 mL of a solution containing 1×10⁻⁶ cells per 5 mL of hydrogel. 8 Culture medium for HUCMSC cells.

[0030] In a second aspect, the present invention provides a 3D-printed liver organoid scaffold for assembling mesenchymal stem cells using FH1 sodium alginate microspheres prepared according to the method.

[0031] Thirdly, the present invention also provides the application of the 3D-printed liver organoid scaffold in the preparation of products that promote the differentiation of functional stem cells.

[0032] The 3D-printed liver organoid scaffold prepared by the method of the present invention can significantly enhance the differentiation of human umbilical cord mesenchymal stem cells into functional stem cells and can be used to construct human liver organoid models. Attached Figure Description

[0033] Figure 1 The structure and composition characterization of FH1@SA microspheres are shown. (A) Schematic diagram of FH1@SA microsphere preparation device and chip; (B) SEM image of FH1@SA microspheres; (C) Micrographs of FH1@SA microspheres produced at different flow rate ratios; (D) Diameter distribution diagram of FH1@SA microspheres produced at different flow rate ratios.

[0034] Figure 2 The physicochemical properties of the hydrogel scaffold materials are shown. (A) Infrared spectra of Gel and GelMA; (B) Infrared spectra of HA and HAMA; (C) 1H NMR spectra of Gel and GelMA; (D) 1H NMR spectra of HA and HAMA. NMR spectrum; (E) Relationship between storage modulus, loss modulus and frequency of HAMA / GelMA hydrogel; (F) Relationship between storage modulus, loss modulus and time of HAMA / GelMA hydrogel; (G) Stress-strain curve of HAMA / GelMA hydrogel; (H) Compressive strength of HAMA / GelMA hydrogel (strain at 30%); (I) Compression image of HAMA / GelMA hydrogel; (J) Swelling curve of HAMA / GelMA / FH1@SA hydrogel in PBS; (K) Degradation curve of HAMA / GelMA / FH1@SA hydrogel in PBS; (L) Degradation curve of HAMA / GelMA / FH1@SA hydrogel in collagenase; (M) Printed image of liver lobule sample; (N) Release curve of FH1; (O) SEM image of hydrogel.

[0035] Figure 3Cytotoxicity was demonstrated. (A) Cytotoxicity of FH1@SA hydrogel microspheres to HUCMSC cells; (B) Cytotoxicity of FHI@SA hydrogel microspheres to HepaRG cells; (C) Cytotoxicity of 3D-printed hydrogel containing HUCMSCs.

[0036] Figure 4 The results of staining for live and dead cells are shown. Live and dead cell images of HUCMSC cells are presented using 3D-printed scaffolds from each group of materials.

[0037] Figure 5 The cytotoxicity results are shown. (A) The effect of each group of 3D printed scaffolds on cell tube formation in HUVEC cells; (B) HUVEC cell tube formation indicators: Nb branches, Nb junctions, and total branching length.

[0038] Figure 6 The results of in vitro stem cell differentiation qPCR detection are shown. The results of qPCR detection of ALB, AFP, CK18, HNF-1α, FOXA2, PRKACA, PRKACB, and PRKX genes in HUCMSCs within an (AH) 3D-printed scaffold are also presented. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0040] Unless otherwise specified, all instruments and reagents used in the embodiments of this specification are conventional instruments and reagents in the art and are commercially available products. Unless otherwise specified, all specific experimental operations involved in this document are understandable or known to those skilled in the art based on their common knowledge or conventional technical means, and will not be described in detail here.

[0041] 1. Preparation Example

[0042] 1.1 Preparation of CaO2@ZIF-8@SL

[0043] CaO2 (32.3 mg) and 2-methylimidazole (3.2 g) were added to 13 mL of water, and Zn(CH3COO)2 (81 mg) and SL (sodium lignosulfonate) (10 mg) were added to 400 mL of water. After mixing all the ingredients, the mixture was incubated overnight at room temperature. The solid was harvested and washed with deionized water, then dried at room temperature to obtain a yellow solid, CaO2@ZIF-8@SL.

[0044] 1.2 Preparation of FH1-loaded sodium alginate microspheres (FH1@SA)

[0045] Mix 1.2 mL of 2M calcium chloride (CaCl2) with 4.8 mL of 0.5M ethylenediaminetetraacetic acid (EDTA) solution until homogeneous. Then add 2M sodium hydroxide solution to adjust the pH to about 7.2. Finally, add ultrapure water to make up to 10 mL to obtain Ca-EDTA solution.

[0046] Weigh 10 mg of sodium alginate (SA) and dissolve it in 0.5 mL of deionized water. Place the solution at 60 °C until completely dissolved. Dissolve 1 mg of FH1 (CAS No. 2719-05-3, BRD-K4477, purity 99.8%, Selleck) in 25 μL of DMSO. Mix the FH1 solution with the SA solution to obtain an FH1@SA solution.

[0047] Add an equal volume of Ca-EDTA solution to the above FH1@SA solution and mix well to obtain FH1@SA / Ca-EDTA solution.

[0048] The FH1@SA / Ca-EDTA solution and 2% microdroplet-generated oil were separately loaded into 2.5 mL syringes and fed into a microinjection pump. The flow rate of the aqueous phase (FH1@SA / Ca-EDTA solution) was set to 0.1 mL / h, and the flow rate of the oil phase (2% microdroplet-generated oil) was set to 1 mL / h. Microspheres were formed by extrusion through a microfluidic chip. The generated microdroplets were collected using a centrifuge tube containing the oil phase of 1% acetic acid, and then allowed to solidify for 30-60 min to form hydrogel microspheres.

[0049] Then, the oil generated from the bottom microdroplets was removed, and a demulsifier with a volume twice that of the microspheres was added. The mixture was centrifuged at 1000 rpm for 30 seconds, and the bottom demulsifier was removed. Finally, the microspheres were washed with PBS 2-3 times to collect the hydrogel microspheres.

[0050] 1.3 Preparation of Methacrylamide Gelatin (GelMA)

[0051] First, 5.0 g of gelatin was weighed and added to 50 mL of PBS buffer. The solution was dissolved completely with magnetic stirring in a 50°C water bath until fully transparent. Then, 3 mL of methacrylic anhydride was slowly added dropwise to the gelatin solution, and the mixture was magnetically stirred at 700 rpm for 1 hour, maintaining the reaction temperature at 50°C. The reaction solution contained a large number of oily droplets. After the reaction was complete, the solution was transferred to a cellulose dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water at 40°C for 3 days to remove byproducts. The dialyzed reaction solution was collected, centrifuged at 5000 rpm for 10 minutes to remove the precipitate, and the supernatant was collected and freeze-dried at -80°C to obtain the final product, GelMA.

[0052] 1.4 Preparation of Methacrylamide Hyaluronic Acid (HAMA)

[0053] Weigh 5.0 g of hyaluronic acid and dissolve it in 400 mL of deionized water, stirring mechanically until completely dissolved. Add 12 mL of methacrylic anhydride, adjust the pH of the reaction solution to 8.5 with 5 M NaOH, stir at room temperature for 24 hours, then dialyze the reaction using a cellulose dialysis bag with a molecular weight cutoff of approximately 1.2 kDa, and freeze-dry to obtain HAMA.

[0054] 1.5 3D Printing of GelMA / HAMA Loaded FHI@SA Microspheres and CaO2@ZIF-8@SL Nanoparticles

[0055] GelMA and HAMA bio-ink were prepared by dissolving methacryloyl gelatin (GelMA) and methacryloyl hyaluronic acid (HAMA) in deionized water containing a certain proportion of LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphonate, a photoinitiator) and tartrazine (a light blocker that helps print small pores).

[0056] First, prepare the photoinitiator solution by adding 1 mg of LAP and 0.6 mg of lemon yellow to 1 mL of deionized water and vortexing for 30 seconds to mix thoroughly. Dissolve 0.01 g of HAMA in 1 mL of deionized water to obtain a 1% HAMA solution. Dissolve a certain amount (0, 0.025, 0.050, 0.075 g) of GelMA in the HAMA solution to obtain a GelMA / HAMA mixed solution. Then, add 5 mg of CaO2@ZIF-8@SL and 2 mg of FHI@SA complex. Next, pour the prepared bio-ink into the printer, select the printing model and size, and print (light intensity: 18 mW / cm²; exposure time: 24 s; number of substrate layers: 1; substrate exposure time: 26 s). Finally, wash the printed hydrogel 2-3 times with sterile PBS to obtain the hydrogel scaffold.

[0057] 1.6 3D printing of GelMA / HAMA / HUCMSCs loaded with FHI@SA microspheres and CaO2@ZIF-8@SL nanoparticles

[0058] First, prepare the photoinitiator solution by adding 1 mg of LAP and 0.6 mg of lemon yellow to 1 mL of deionized water and vortexing for 30 seconds to mix thoroughly. Dissolve 0.01 g of HAMA in 1 mL of deionized water to obtain a 1% HAMA solution. Dissolve a certain amount (0, 0.025, 0.050, 0.075 g) of GelMA in the HAMA solution to obtain a GelMA / HAMA mixed solution. Then add 5 mg of CaO2@ZIF-8@SL and 2 mg of FHI@SA complex to obtain a hydrogel solution. Take 5 mL of the hydrogel solution and add 1 mL of solution containing 1×10⁻⁶ ppm of HAMA. 8Bioink was obtained by culturing HUCMSC cells in α-MEM complete medium.

[0059] Then, the prepared bio-ink is poured into the printer, the printing model and size are selected, and the print is completed (light intensity: 18mW / cm2; exposure time: 24s; number of base layers: 1; base layer exposure time: 26s). Finally, the printed hydrogel is washed 2-3 times with sterile PBS to obtain the hydrogel scaffold.

[0060] 1.7 3D Printing of GelMA / HAMA Loaded FHI@SA Microspheres

[0061] The preparation steps are similar to those in “1.6 3D printing of GelMA / HAMA / HUCMSCs loaded with FHI@SA microspheres and CaO2@ZIF-8@SL nanoparticles”, except that CaO2@ZIF-8@SL nanoparticles are not added.

[0062] 2. Morphology and size distribution of microspheres

[0063] A 1% FH1@SA / Ca-EDTA solution was used as the dispersed phase. The flow rate ratio of the dispersed phase to the mobile phase was controlled at 1:2.5, 1:5, and 1:10, respectively. Microspheres were prepared using a microfluidic device, and the morphology of the microspheres was photographed under a microscope. The microsphere size was also statistically analyzed using ImageJ.

[0064] The collected microspheres were freeze-dried, then attached to conductive adhesive. Gold was then sputtered onto the hydrogel surface for 60 seconds. The surface morphology of the 3D-printed hydrogel was observed using a scanning electron microscope. The test conditions were: 5kV electron beam.

[0065] Devices and chips for preparing microspheres, such as Figure 1 The installation is shown as A in the diagram. Figure 1 Image B in the image is a SEM image of the microspheres. As can be seen from the image, the surface of the microspheres has a certain porous structure. The morphology of the prepared microspheres can be observed under a microscope, such as... Figure 1 As shown in Figure C, FH1-loaded sodium alginate microspheres were successfully prepared using microfluidic technology, exhibiting uniform and controllable size. Furthermore, the size of the microspheres was quantitatively analyzed. Figure 1 As shown in Figure D), the microsphere sizes prepared at dispersed phase / mobile phase flow rates of 1:2.5, 1:5, and 1:10 were 260.2 μm, 225.2 μm, and 210.1 μm, respectively. The microsphere size gradually decreased with decreasing flow rate. This indicates that a faster mobile phase flow rate results in greater fluid shear force on the dispersed phase upon entering the mobile phase. This force promotes the emulsification process of droplets, accelerates the breakup of droplets into spheres, and thus results in smaller and more numerous microspheres formed per unit time.

[0066] 3. Structural composition and physicochemical characterization of hydrogels

[0067] 3.1 Infrared

[0068] Weigh 8 mg of the sample to be tested and an appropriate amount of dry potassium bromide powder (mass ratio of about 5%) into an agate mortar. Grind thoroughly to mix the two evenly. Then take an appropriate amount of the ground sample powder and press it into a tablet (vacuum pressure 20 mmHg, pressing for 5 min) to obtain a sample thin film. Set the scanning range to 4000-400 cm-1 and use a Fourier transform infrared spectrometer for detection.

[0069] from Figure 2 As can be seen from A in GelMA, at 1632cm -1 1532cm -1 1231cm -1 The characteristic peaks of amide I, amide II, and amide III on the Gel were significantly stronger than the corresponding characteristic peaks of Gel, indicating that methacrylic acid groups were introduced into the Gel molecular chain, generating new amide bonds, thus demonstrating the successful synthesis of GelMA.

[0070] from Figure 2 As can be seen from B, HAMA is at 1710cm. -1 1630cm -1 The appearance of new characteristic peaks is due to the ester bonds in methacrylic anhydride, indicating that methacrylic acid groups were successfully introduced into the HA molecule, signifying the successful synthesis of HAMA.

[0071] 3.2 Nuclear Magnetic Resonance Imaging

[0072] Weigh 8 mg of the sample to be tested, add deuterated heavy water to dissolve, and sonicate until the solution is completely dissolved and clear. Then, put it into a clean NMR tube and use a nuclear magnetic resonance spectrometer to determine the NMR structure at room temperature. The spectrum is then analyzed using MestReNova software.

[0073] Figure 2 C in the figure represents the 1H NMR spectrum of Gel and GelMA. Analysis of the NMR results revealed new acrylic acid proton peaks (=CH2) at 5.35 and 5.58 ppm, which correspond to the absorption peaks of hydrogen on the methacrylamide olefin bond, further proving the successful modification of Gel.

[0074] Figure 2 D in the figure represents the 1H NMR spectrum of HA and HAMA. Analysis of the NMR results revealed new peaks at 6.10 and 5.66 ppm, which are attributed to vinyl peaks in methacrylic acid, further confirming the successful modification of HA.

[0075] 3.3 Rheological Testing

[0076] Rheological measurements were performed using a stainless steel parallel plate rotor with a diameter of 25 mm. G' represents the storage modulus of the sample, and G" represents the loss modulus of the sample. Dynamic strain scanning was performed at room temperature from 0.1 to 100 rad / s to determine the linear viscoelastic range of the hydrogel, and the changes in storage modulus (G') and loss modulus (G") were recorded.

[0077] Figure 2 Figures E and F illustrate the relationship between the storage modulus (G') and loss modulus (G”) of the hydrogel and frequency and time, respectively. When G' is greater than G”, the hydrogel exhibits a gel state. The figure shows that after gelation, G' remains greater than G” with increasing time and angular frequency, indicating that the hydrogel forms a stable three-dimensional network structure, thus maintaining its gel state. Furthermore, G' continuously increases with increasing GelMA concentration, suggesting that the elastic properties of the hydrogel scaffold gradually improve with increasing GelMA concentration, demonstrating that the addition of GelMA effectively enhances the elastic properties of the hydrogel scaffold. This may be because: with increasing GelMA concentration, the crosslinking density also increases, forming more crosslinking points between GelMA chains, resulting in a stronger network structure. Secondly, with increasing GelMA concentration, the intermolecular forces also strengthen, making the crosslinked network of the hydrogel more compact and improving its elastic properties.

[0078] 3.4 Compression Modulus

[0079] 500 μL of hydrogel was prepared into a cylindrical shape with uniform height and a base diameter of 11 mm. The prepared hydrogel sample was then placed on the sample stage of a universal testing machine, and the clamp height was adjusted so that the top and bottom surfaces of the hydrogel were just in contact with the clamp. The compression rate was then set to 0.05 mm / s and compressed at a constant rate until 70% of the sample strain was reached. The pressure and displacement values ​​were recorded. The compressive modulus was calculated using the following formula:

[0080]

[0081] Where F is the recorded pressure value and S is the cross-sectional area of ​​the hydrogel sample.

[0082] Figure 2In the figures, G, H, and I represent the stress-strain curve, compressive strength at 30% deformation, and compression image of the hydrogel, respectively. The hydrogel with 1% H fractures easily under light pressure and cannot return to its original shape. The hydrogel with 7.5% GelMA can resist certain pressure and returns to its original shape after compression, demonstrating that the addition of GelMA improves the mechanical properties of the hydrogel. The stress-strain curves show that the compressive strength of the hydrogel increases with increasing GelMA concentration. At 30% deformation, the compressive strength of the hydrogel with 1% H is 0.37 ± 0.07 kPa, the compressive strength with 1% HAMA / 2.5% GelMA is 4.47 ± 0.10 kPa, and the compressive strength with 1% HAMA / 5% GelMA is 9.22 ± 0.09 kPa. The compressive strength of 1% HAMA / 7.5% GelMA is 12.55±0.86 kPa. This is likely because a higher concentration of GelMA results in a tighter cross-linked network and stronger intermolecular forces, thus providing greater resistance to external forces and superior mechanical properties. Hydrogel moduli that match the elastic properties of the liver are around 1-10 kPa. Considering rheological properties, combinations of 1% HAMA / 5% GelMA and 1% HAMA / 7.5% GelMA can be selected as hydrogel formulations for treating liver injury, as they are more conducive to liver recovery.

[0083] 3.5 In vitro swelling of hydrogel

[0084] Add 500 μL of hydrogel precursor solution to each well of a 48-well plate. After photocrosslinking, remove the sample and weigh it on a plastic petri dish, recording the initial weight of the hydrogel as W0. Place the prepared hydrogel sample in a 24-well plate and add PBS buffer using a pipette to completely submerge the sample. At intervals of 1, 2, 4, 6, 8, 12, and 24 hours, remove the sample, carefully wipe off excess moisture with weighing paper, weigh it on a plastic petri dish, and record the weight as W0. t .

[0085] The swelling ratio is calculated using the following formula:

[0086]

[0087] Hydrogels used for liver injury treatment should have a moderate swelling rate, enabling rapid water absorption in the initial stages of treatment without excessive swelling leading to structural collapse. Figure 2As can be seen from the J diagram, increasing the GelMA concentration leads to a decrease in the swelling rate of the hydrogel. This is because increased GelMA concentration enhances the cross-linking density between polymer chains, resulting in a tighter cross-linked network that allows for more effective water absorption, thus limiting the swelling of the hydrogel scaffold. The addition of FH1-loaded sodium alginate microspheres (FH1@SA) significantly increased the swelling rate of the hydrogel, reaching 200%-250%. This is because sodium alginate is a highly hydrophilic polysaccharide; its addition improves the overall hydrophilicity of the hydrogel, promoting water molecule penetration and thus increasing the swelling rate. Furthermore, the introduction of FH1@SA may increase the porosity and permeability of the hydrogel network, making it easier for water molecules to enter the hydrogel interior. In conclusion, altering the GelMA concentration and introducing FH1@SA can regulate the swelling properties of the hydrogel scaffold, giving the hydrogel good hydration properties for rapid water absorption and retention in the in vivo environment. This helps provide a suitable microenvironment, promoting the survival and regeneration of hepatocytes.

[0088] 3.6 In vitro degradation of hydrogels

[0089] Add 500 μL of hydrogel precursor solution to each well of a 48-well plate. After photocrosslinking, remove the plate and freeze-dry at -80°C. Weigh the sample and record the initial weight of the freeze-dried hydrogel as W0. Place the prepared freeze-dried hydrogel sample in a 24-well plate and add a fixed volume of PBS buffer or 5 U / mL collagenase II solution using a pipette to completely submerge the hydrogel sample. Place the plate in a 37°C shaker. Replace the degradation solution every 3 days. At intervals of 3, 7, 10, 14, 21, and 28 days, remove the sample, discard excess degradation solution, freeze-dry, weigh, and record the weight of the hydrogel as W0. t The degradation rate is calculated using the following formula:

[0090]

[0091] The ideal degradation rate of liver injury repair materials should match the rate of new liver tissue regeneration. If it is too slow, it will hinder the formation of new liver tissue; if it is too fast, it will lose its supporting function.

[0092] Figure 2In the figure, K and L represent the degradation performance of the hydrogel in PBS and collagenase II, respectively. As can be seen from the figure, the degradation rate of the hydrogel increased after the addition of sodium alginate microspheres in both PBS and collagenase II. This may be because sodium alginate has strong water absorption, increasing the overall water absorption of the hydrogel, leading to hydrolysis or enzymatic degradation of the hydrogel's network structure. Secondly, the addition of sodium alginate microspheres may affect the degree of cross-linking of the hydrogel, reducing the cross-linking density. Simultaneously, the pores on the surface of the sodium alginate microspheres can facilitate the entry of water molecules and enzymes into the hydrogel, accelerating the degradation rate.

[0093] 3.7 Appearance and compression fracture images of the hydrogel scaffold

[0094] The hydrogel scaffold was observed under white light using a camera to examine its front mesh structure. Furthermore, a 10mm high hydrogel was compressed, and its cracking was observed as compression progressed.

[0095] The printing model used is a liver lobule model, from Figure 2 As can be seen from M, this material can print complete liver lobule models, the printed hydrogel structure is clear and has certain support properties, and the printability is good.

[0096] 3.8FH1 in vitro drug release

[0097] Weigh 100 mg of FH1@SA and sonicate it thoroughly with 1 mL of 7.5% GelMA / 1% HAMA solution, then crosslink it under light. Transfer the mixture to a centrifuge tube and add 2 mL of PBS buffer (containing 0.1% Tween 80). At days 1, 3, 7, 10, 14, 21, and 28, collect 2 mL of the sustained-release solution and add 2 mL of fresh PBS buffer (containing 0.1% Tween 80). Measure the absorbance of the drug in the sustained-release solution using a UV spectrophotometer, calculate the drug concentration based on the standard curve, and plot the release curve.

[0098] The formula for calculating drug release is as follows:

[0099]

[0100] E r Cumulative drug release

[0101] V e The volume of each sample taken (i.e., 2 ml).

[0102] C i The drug concentration measured during the i-th sampling.

[0103] m drug Total mass of the drug

[0104] FH1 release in the stent as follows Figure 2 As shown in N, compared to the HAMA / GelMA / FH1 hydrogel scaffold, the HAMA / GelMA / SA@FH1 scaffold exhibits slower release. This may be because FH1 is encapsulated within SA microspheres, and SA can control the release of FH1. As SA degrades, FH1 is gradually released as well. The SA microspheres possess a certain porosity, and encapsulating FH1 within them forms a sustained-release system, allowing for the gradual release of FH1 after scaffold implantation. This sustained-release property is beneficial for continuously stimulating hepatocytes and promoting regeneration over a long period.

[0105] 3.9SEM

[0106] After freeze-drying the prepared hydrogel, the surface and cross-section (quenched with liquid nitrogen) portions were attached to conductive adhesive. Gold was then sprayed onto the hydrogel surface for 60 seconds, and the chamber was placed in a vacuum chamber. The microstructure of the surface and cross-section of the 3D-printed hydrogel was observed using a scanning electron microscope. The test conditions were: 5kV electron beam.

[0107] Figure 2 O in the image represents the SEM image of the hydrogel. The image shows that the hydrogel has a cross-linked network structure with high porosity and many particulate substances attached to it, indicating that the hydrogel successfully encapsulates hydrogel microspheres.

[0108] 4. Cell Experiment

[0109] 4.1 Cytotoxicity of FH1@SA hydrogel microspheres

[0110] After the FH1@SA powder was irradiated with UV overnight, a 10 mg / mL FH1@SA solution was prepared with sterile PBS. The FH1@SA solution was then diluted with DMEM complete medium to concentrations of 5, 2.5, 0.5, 0.25, 0.1, 0.05, and 0.025 mg / mL for later use.

[0111] HUCMSC cells and HepaRG cells were digested with 0.05% trypsin. HUCMSC cells were resuspended in α-MEM complete medium, and HepaRG cells were resuspended in DMEM complete medium. Cells were seeded at a density of 5000 cells / well in 96-well plates and cultured at 37°C in a CO2 incubator (containing 5% CO2) for 24 h until cell adhesion was achieved. The medium was then aspirated, the cells were washed with PBS, and different concentrations of FH1@SA solution were added for further culture for 24 h. After culture, the medium was aspirated, the cells were washed three times with PBS, and 100 μL of CCK-8 working solution was added to each well. The cells were incubated at 37°C in a CO2 incubator (containing 5% CO2) for 30 min. The absorbance (OD) was measured at 450 nm using a microplate reader, and cell viability was calculated using the following formula:

[0112]

[0113] The effect of different concentrations of FHI@SA hydrogel microspheres on the viability of HUCMSCs was evaluated using the CCK-8 assay. Figure 3 As shown in Figure A, the activity of HUCMSC cells reached a maximum of 107.19% at a drug concentration of 0.5 mg / mL, and the activity of HUCMSC cells did not change significantly at other concentrations. This indicates that the FHI@SA hydrogel microsphere solution does not produce toxic side effects on HUCMSC cells.

[0114] The effect of different concentrations of FHI@SA hydrogel microsphere solutions on the viability of HepaRG cells was evaluated using the CCK-8 assay. Figure 3 As shown in Figure B, the activity of HepaRG cells reached a maximum of 107.60% at a drug concentration of 0.5 mg / mL, and the activity of HepaRG cells did not change significantly at other concentrations. This indicates that the FHI@SA hydrogel microsphere solution does not produce toxic side effects on HepaRG cells.

[0115] 4.2 Cytotoxicity of 3D-printed hydrogels containing HUCMSCs

[0116] HUCMSC cells were digested with 0.05% trypsin, and 1×10⁻⁶ cells were added. 8 Each cell was resuspended in 1 mL of α-MEM complete medium. The α-MEM complete medium containing cells was then mixed with 5 mL of each group of hydrogel solutions (GelMA / HAMA, GelMA / HAMA / FH1@SA, GelMA / HAMA / CaO2@ZIF-8@SL, GelMA / HAMA / FH1@SA / CaO2@ZIF-8@SL). These solutions were then 3D printed into 8×8×1.5 mm hydrogel scaffolds (GHH, GHHF, GHHC, GHHFC) and cultured in α-MEM complete medium. The scaffolds were placed in 48-well plates and cultured for 24, 72, and 120 h, respectively. After culture, the culture medium was discarded, and the cells were washed three times with PBS. 100 μL of CCK-8 working solution (α-MEM basal medium containing 10% CCK-8) was added to each well. The cells were incubated in a 37°C CO2 incubator for 30 min. The hydrogel scaffolds of each group were then broken, centrifuged, and the supernatant was transferred to a 96-well plate. The absorbance (OD) was measured at 450 nm using a microplate reader. Cell viability was calculated using the following formula:

[0117]

[0118] The CCK-8 assay was used to evaluate the effects of each group of materials on the proliferation and viability of HUCMSC cells. Figure 3 As shown in Figure C, the survival rate of HUCMSCs gradually increased with prolonged culture time (Day 1, Day 3, Day 5), indicating that the material had no significant cytotoxicity. Notably, the GHHFC group exhibited a significant proliferative effect: by day 3, its cell activity was significantly higher than other groups (p<0.05), and by day 5, the differences between groups further widened, with the GHHFC group showing the most significant increase in HUCMSC cell activity, suggesting that it has a better effect on promoting cell growth.

[0119] 4.3 Staining of live and dead cells

[0120] HUCMSC cells were digested with 0.05% trypsin, and 1×10⁻⁶ cells were added. 8 Cells were resuspended in 1 mL of α-MEM complete medium. The α-MEM medium containing cells was then mixed with 5 mL of each group of hydrogel solutions (GelMA / HAMA, GelMA / HAMA / FH1@SA, GelMA / HAMA / CaO2@ZIF-8@SL, GelMA / HAMA / FH1@SA / CaO2@ZIF-8@SL). These solutions were then 3D printed into 8×8×1.5 mm hydrogel scaffolds (GHH, GHHF, GHHC, GHHFC) and cultured at 37°C in a CO2 incubator (containing 5% CO2) for 24, 72, and 120 h. The medium was then aspirated, and the cells were washed three times with PBS. Cell viability and mortality were then added to the cell viability and mortality staining working solution (2 μM calcein AM, 8 μM PI) and incubated at room temperature for 20 min. Immediately after washing three times with PBS, images were acquired using a fluorescence microscope to analyze cell viability and mortality. Calcein AM labels live cells and emit green fluorescence, while propidium iodide (PI) labels dead cells and emit red fluorescence.

[0121] Figure 4 The effects of different 3D-printed hydrogel scaffolds on the survival of HUCMSCs were assessed using live and dead cell staining. Compared with the control group, no significant PI positive signal (dead cells) was observed in any of the experimental groups, indicating that the materials have good biocompatibility and no significant cytotoxicity. Notably, the CHHFC group showed a significantly enhanced Calcein-AM positive signal (live cells), with denser cell distribution and better proliferation status.

[0122] 4.4 In vitro angiogenesis

[0123] First, thawed Matrigel was spread evenly at a rate of 100 μL / well onto a 48-well plate and incubated in a cell culture incubator for 30 min to allow it to transition from a liquid to a solid gel. HUVECs were cultured at a density of 1 × 10⁻⁶ cells / well. 5Cells were seeded at a density of / well on a Matrigel surface, and Transwell chambers were added, with cells in the lower chamber and 3D-printed scaffolds in the upper chamber. α-MEM complete culture medium was added. After culturing at 37°C for 6 hours, the tubular network structure formed by the cells was observed under a microscope, and ImageJ software was used to calculate the network structure parameters, including the number of nodes, the number of grids, and the number of trunks.

[0124] Figure 5 Image A shows a fluorescent image of HUVEC cells forming tubular networks, where GHHFC-treated human umbilical cord endothelial cells (HUVECs) form a denser tubular network. Figure 5 B in the figure represents the statistical graph of tube formation indicators. After treatment with GHHFC, the total branch length and number of nodes of HUVECs cells were significantly different from those of the control group (p<0.001), indicating that GHHFC material can effectively promote angiogenesis.

[0125] 4.5 In vitro stem cell differentiation induction

[0126] To induce hepatic differentiation, third-generation HUCMSCs were grouped as described above, printed with bio-ink, and then cultured for 2 weeks in DMEM / F-12 medium containing 0.5 μmol / L dexamethasone, 10 ng / mL epidermal growth factor (Peprotech), 20 ng / mL HGF (hepatocyte growth factor; Peprotech), and 1% ITS (insulin-transferrin-selenium; Invitrogen). Subsequently, the medium was replaced with DMEM / F-12 medium containing 0.5 μmol / L dexamethasone, 20 ng / mL HGF, 10 ng / mL tumor suppressor M (Peprotech), and 1% ITS, and cultured for another 2 weeks.

[0127] 4.5.1 RT-qPCR of ALB, AFP, Ck18 mRNA and GAPDH levels in hepatocytes

[0128] Cells were collected, and the expression of hepatocyte line markers (including AFP, CK-18, ALB, and GAPDH) was detected by real-time RT-qPCR. The cycling conditions were: 95℃ for 10 min, followed by 40 cycles, each cycle consisting of 95℃ for 10 s, 60℃ for 20 s, and 72℃ for 15 s.

[0129] The primers used are designed as follows (forward and reverse):

[0130] ALB (albumin):

[0131] 5'-AGAGGTCTCAAGAAACCTAGGAAA-3'; 5'-GGTTCAGGACCACGGATAGA-3'.

[0132] AFP:

[0133] 5'-TGCAAACGATGAAGCAAGAG-3'; 5'-AACAGGCCTGAGAAATCTGC-3'.

[0134] CK18:

[0135] 5'-TGATGACACCAATATCACACGA-3'; 5'-CTGGGCTTGTAGGCCTTTTA-3'.

[0136] GAPDH (glyceraldehyde-3-phosphate dehydrogenase):

[0137] 5'-ACACCCACTCCTCCACCTTT-3'; 5'-TTACTCCTTGGAGGCCATGT-3'.

[0138] The detailed steps are as follows:

[0139] (1) Extraction of total RNA

[0140] 1) Sample processing: Centrifuge to collect cells into a 1.5 mL centrifuge tube, add 1 mL RNAiso Plus, vortex to mix, and let stand at room temperature for about 5 min.

[0141] 2) Phase separation: Add 0.2 mL of chloroform to each 1 mL of RNAiso Plus, vortex to mix for 15 s, let stand at room temperature for about 3 min, then centrifuge at 12000 rpm for 10 min at 4 ℃.

[0142] 3) Precipitation: Transfer the aqueous phase to a new 1.5 mL centrifuge tube, add 0.5 mL of isopropanol to each 1 mL of RNAiso Plus, mix well, let stand at room temperature for 10 min, then centrifuge at 12000 rpm for 10 min at 4 °C.

[0143] 4) Washing: Discard the supernatant, add 1 mL of 75% ethanol to each 1 mL of RNAiso Plus, mix well, and centrifuge at 7500 rpm for 5 min at 4°C.

[0144] 5) Dissolve: Discard the supernatant and air-dry the RNA precipitate for about 5 minutes (note that it should not be completely dried, just until the precipitate turns white). Add an appropriate amount of DEPC-treated water to dissolve the RNA precipitate.

[0145] 6) Determine concentration and purity: Record the data after determining the RNA concentration and purity using a spectrophotometer.

[0146] (2) First-strand cDNA synthesis:

[0147] 1) Prepare the reverse transcription reaction solution according to the following composition. The preparation of the reaction solution should be carried out on ice.

[0148]

[0149] 2) The reaction was carried out according to the following procedure: 25℃, 5min; 42℃, 30min; 85℃, 5min.

[0150] 3) After the reaction is complete, dilute the product 10 times and store it at -20℃ for later use.

[0151] (3) Quantitative PCR detection

[0152] 1) Dissolve the Mix at 4°C, gently invert to mix, and briefly centrifuge.

[0153] 2) Prepare the reaction solutions shown in the table below on ice.

[0154] Element Added amount Final concentration SYBR Green Master Mix 5μL 1×Mix Forward Primer (2μM) 1μL 0.2μM Reverse Primer (2μM) 1μL 0.2μM cDNA 3μL RNase-free Water to 10μL

[0155] 3) Briefly centrifuge the reaction tube to ensure that all reaction liquid is at the bottom of the reaction well.

[0156] 4) The reaction is carried out using the following procedure.

[0157]

[0158] 4.5.2 RT-qPCR detection of liver-specific transcription factors Hnf-1α and Foxa2

[0159] Cells were collected, and the expression of hepatocyte line markers (including Hnf1α and Foxa2) was detected by real-time RT-qPCR. The cycling conditions were: 95℃ for 10 min, followed by 40 cycles, each cycle consisting of 95℃ for 10 s, 60℃ for 20 s, and 72℃ for 15 s.

[0160] The primers used are designed as follows (forward and reverse):

[0161] Hnf1α: 5'-GACATGCTTCAGTGGGAGAA-3' and 5'-GAGCTGTCTGAGGAGCTGTG-3'.

[0162] Foxa2: 5'-TCGGAGGAGGAGGAGGAGAG-3' and 5'-CGGAGGAGGGAGGAGGAGAG-3'.

[0163] 4.5.3 RT-qPCR detection of hepatocyte maturation-related genes Prkaca, Prkacb, and Prkx

[0164] Cells were collected, and the expression of hepatocyte line markers (including Prkaca, Prkacb, and Prkx) was detected by real-time RT-qPCR. The cycling conditions were: 95℃ for 10 min, followed by 40 cycles, each cycle consisting of 95℃ for 10 s, 60℃ for 20 s, and 72℃ for 15 s.

[0165] The primers used are designed as follows (forward and reverse):

[0166] Prkaca: 5'-ATGGAGGACAGGAGAGGAGG-3' and 5'-AGGAGGGAGGAGGAGGAGG-3'

[0167] Prkacb: 5'-ATGAGGAAGAGGAGGAGGA-3' and 5'-AGGAGGAGGAGGAGGAAGG-3'

[0168] Prkx: 5'-ATGGAGGAGGAGGAGGAAGG-3' and 5'-AGGAGGAGGAGGAGGAGGA-3'.

[0169] 4.5.4 Immunofluorescence staining of liver maturation markers ALBUMIN and Zonula Occludens-1 protein

[0170] To detect the liver maturation markers ALBUMIN and Zonula Occludens-1 protein, third-generation HUCMSCs were grouped as described above, printed with bio-ink, and then cultured for 2 weeks in DMEM / F-12 medium containing 0.5 μmol / L dexamethasone, 10 ng / mL epidermal growth factor (Peprotech), 20 ng / mL HGF (hepatocyte growth factor; Peprotech), and 1% ITS (insulin-transferrin-selenium; Invitrogen). Subsequently, the medium was replaced with DMEM / F-12 medium containing 0.5 μmol / L dexamethasone, 20 ng / mL HGF, 10 ng / mL tumor suppressor M (Peprotech), and 1% ITS, and cultured for another 2 weeks.

[0171] Fix each group of hydrogel scaffolds with 1% paraformaldehyde for 15 min, and wash 1-2 times with PBS. Add permeabilization buffer and permeabilize for 10 min, followed by blocking with 1% BSA. Wash 1-2 times with PBS, add primary antibody working solution, and incubate for 1 h. Wash 1-2 times with PBS, add fluorescent secondary antibody working solution, and incubate for 1 h. Wash 1-2 times with PBS, add anti-fluorescence quencher containing DAPI, and mount. Observe and photograph under a microscope.

[0172] qPCR analysis showed that the GHHFC group had the most significant promoting effect on the differentiation of HUCMSCs into hepatocytes: compared with the GHH group, the GHHFC group significantly upregulated the expression of mature hepatocyte markers ALB (2.13-fold), AFP (1.78-fold), and CK18 (1.79-fold). Figure 6 The presence of AC in the data indicates that it can effectively promote the functional maturation of hepatocytes. Simultaneously, the expression of key regulatory factors for hepatocyte development, HNF-1α (2.26-fold) and FOXA2 (2.54-fold), was also significantly enhanced. Figure 6 The presence of D and E in the data suggests that GHHFC may function by activating hepatocyte differentiation. Furthermore, the expression of genes closely related to hepatocyte metabolism, PRKACA (2.1-fold), PRKACB (2.5-fold), and PRKX (2.18-fold), were all significantly upregulated. Figure 6 The presence of FH in the study further confirms that GHHFC can promote the functional maturation of hepatocytes. These results indicate that GHHFC significantly enhances the ability of HUCMSCs to differentiate into functional hepatocytes by regulating key hepatocyte differentiation factors and metabolism-related genes.

[0173] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for constructing a 3D-printed liver organoid scaffold loaded with FH1 sodium alginate microspheres and assembled with mesenchymal stem cells, comprising the following steps: S1. Preparation of CaO2@ZIF-8@SL; S2. Preparation of sodium alginate microspheres FH1@SA loaded with FH1; S3. Fabrication of 3D-printed liver organoid scaffolds; Step S1 includes: adding CaO2 and 2-methylimidazole to water, mixing with Zn(CH3COO)2 and sodium lignosulfonate SL, incubating overnight, harvesting the solid and washing with deionized water, and then drying at room temperature to obtain CaO2@ZIF-8@SL; the weight ratio of CaO2, 2-methylimidazole, Zn(CH3COO)2 and sodium lignosulfonate is (30-35):(3000-3500):(75-85):(8-12); Step S2 includes: S201, calcium chloride solution and ethylenediaminetetraacetic acid solution are mixed evenly, then the pH is adjusted to 7.2, and finally ultrapure water is added to make up the volume to obtain Ca-EDTA solution; S202. Dissolve 4,4'-diacetamidodiphenylmethane FH1 in DMSO to obtain FH1 solution, then mix it with sodium alginate aqueous solution to obtain FH1@SA solution. S203. Add the FH1@SA solution to an equal volume of Ca-EDTA solution and mix thoroughly to obtain an FH1@SA / Ca-EDTA solution. S204. FH1@SA / Ca-EDTA solution and 2% microdroplet-generating oil are injected into syringes respectively, extruded through a microfluidic chip to form microspheres, and the generated microdroplets are collected and then allowed to solidify to form hydrogel microspheres; then the bottom microdroplet-generating oil is removed, a demulsifier is added, centrifuged and the bottom demulsifier is removed, and finally the microspheres are washed with PBS to collect the FH1-loaded sodium alginate microspheres FH1@SA; In step S2, the mass ratio of methacrylamide gelatin, methacrylamide hyaluronic acid, CaO2@ZIF-8@SL nanoparticles, and FH1@SA is (25-75):10:5:2; Step S3 includes: Methacrylamide gelatin and methacrylamide hyaluronic acid were dissolved in deionized water containing lithium phenyl-2,4,6-trimethylbenzoylphosphonate and tartrazine, and mixed thoroughly. Then, CaO2@ZIF-8@SL and FH1@SA were added and mixed thoroughly to obtain a hydrogel solution. HUCMSC cells were then added to obtain bio-ink. The prepared bio-ink was used for printing and cleaning to obtain a 3D-printed liver organoid scaffold assembled with FH1 sodium alginate microspheres.

2. The method according to claim 1, characterized in that, The methacrylamide gelatin is prepared by the following steps: Methacrylic anhydride was added dropwise to PBS buffer containing gelatin and the mixture was magnetically stirred. After the reaction was complete, the mixture was dialyzed in deionized water to remove byproducts. The dialyzed reaction solution was collected, centrifuged to remove the precipitate, and the supernatant was collected and freeze-dried to obtain the final product, methacrylamide gelatin.

3. The method according to claim 2, characterized in that, The mass ratio of gelatin to methacrylic anhydride is (1-2):

1.

4. The method according to claim 1, characterized in that, The methacrylamide hyaluronic acid was prepared by the following steps: Hyaluronic acid was dissolved in deionized water and mechanically stirred until completely dissolved. Methacrylic anhydride was added, and the pH was adjusted to 8.5 with alkali. The mixture was stirred at room temperature, and then dialyzed and lyophilized to obtain methacrylated hyaluronic acid.

5. The method according to claim 4, characterized in that, The mass ratio of hyaluronic acid to methacrylic anhydride is 1:(1-3).

6. The method according to claim 1, characterized in that, In step S3, the amount of HUCMSC cells added is 1 mL of HUCMSC cells per 5 mL of hydrogel containing 1×10⁻⁶ cells. 8 Culture medium for HUCMSC cells.

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