Construction method of oxygen self-producing biological 3D printing mesenchymal stem cell liver organoid scaffold

Abstract

The invention discloses a construction method of a self-oxygen-production biological 3D printing mesenchymal stem cell liver organoid scaffold. The construction method comprises the following steps: preparing CaO2 (at) ZIF-8 (at) SL nanoparticles; and adding the CaO2 (at) ZIF-8 (at) SL nanoparticles into a mixed solution of methacrylated gelatin and methacrylated hyaluronic acid, so as to obtain the self-oxygen-production biological 3D printing mesenchymal stem cell liver organ scaffold. The self-oxygen-producing biological 3D printing mesenchymal stem cell liver organoid scaffold prepared by the method has the stability of releasing oxygen for a long time, has good ROS (reactive oxygen species) removal capacity, is free of cytotoxicity, and can be used for constructing a human liver organoid model.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of liver organoid construction, and particularly relates to a method for constructing a self-oxygen-producing biological 3D-printed mesenchymal stem cell liver organoid scaffold. BACKGROUND

[0002] In recent years, 3-dimensional (3D) printing technology has been widely used to reconstruct in vitro tissue models using living organisms, i.e., cellular reconstitutes. In liver tissue engineering, studies using 3D-printed liver tissue models have been established, which significantly improve the response and function of cells in drug screening, liver diseases, and liver regeneration medicine. However, due to the complex structure and physiological function of the liver, the use of a single-component bio-ink cannot meet the oxygen (O2) demand of organoid models, and therefore, we need to integrate multi-component bio-inks to provide a favorable microenvironment to promote cell proliferation and differentiation, and even initiate angiogenesis.

[0003] Timely and sufficient release of O2 is crucial for improving cell survival. O2 release materials are generally classified into oxygen-carrying materials and oxygen-generating materials. Oxygen-carrying materials such as natural hemoglobin, perfluorocarbons, and cyclodextrins may cause severe damage to cells within a few hours due to the burst release of O2. In addition, the O2 release time is too short to support 2-4 weeks of vascular reconstruction.

[0004] Suitable scaffold pore sizes can construct a favorable microenvironment for O2 release and vascular regeneration. The ability of hydrogels to embed drugs enables them to be integrated with nanoparticles or cells to achieve multifunctionality. SUMMARY

[0005] The purpose of the present application is to solve the above technical problems, and to provide a method for constructing a 3D-printed mesenchymal stem cell liver organoid scaffold with good mechanical strength and biological activity, and stable oxygen release.

[0006] To achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0007] In a first aspect, the present application provides a method for constructing a self-oxygen-producing biological 3D-printed mesenchymal stem cell liver organoid scaffold, comprising the following steps:

[0008] S1, preparing CaO2@ZIF-8@SL nanoparticles;

[0009] S2, adding the CaO2@ZIF-8@SL nanoparticles into a mixed solution of methacrylated gelatin and methacrylated hyaluronic acid to obtain a hydrogel solution;

[0010] S3, adding HUCMSC cells into the hydrogel solution to obtain a bio-ink, and performing 3D printing to obtain the self-oxygen-producing biological 3D-printed mesenchymal stem cell liver organoid scaffold.

[0011] Preferably, the step S1 comprises: adding CaO2 and 2-methylimidazole into water, mixing with Zn(CH3COO)2 and sodium lignosulfonate, incubating overnight, harvesting the solid and washing with deionized water, and then drying to obtain CaO2@ZIF-8@SL nanoparticles.

[0012] More preferably, in the 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.

[0013] More preferably, the sodium lignosulfonate is in the form of a 2-5% (volume concentration) aqueous sodium lignosulfonate solution.

[0014] Preferably, the step S2 comprises:

[0015] The methacrylated gelatin and the methacrylated hyaluronic acid are dissolved in deionized water containing LAP and tartrazine, mixed uniformly, then the CaO2@ZIF-8@SL nanoparticles are added and mixed uniformly, and then the prepared bio-ink is printed and washed to obtain the self-oxygen-producing biological 3D-printed mesenchymal stem cell liver organoid scaffold.

[0016] Preferably, in the step S2, the mass ratio of the methacrylated gelatin, the methacrylated hyaluronic acid and the CaO2@ZIF-8@SL nanoparticles is (10-15):2:1.

[0017] Preferably, the methacrylated gelatin is prepared by the following steps:

[0018] The methacrylic anhydride is added dropwise to the PBS buffer containing gelatin, and magnetically stirred. After the reaction is completed, the by-products are removed by dialysis in deionized water. The reaction solution after dialysis is collected, centrifuged to remove the precipitate, and the supernatant is collected and freeze-dried to obtain the final product, methacrylated gelatin.

[0019] More preferably, the concentration of the gelatin in the PBS buffer is 0.1-0.5 g / mL, and the most preferred concentration is 0.1 g / mL.

[0020] More preferably, the mass ratio of the gelatin to the methacrylic anhydride is (1-2):1, and the most preferred mass ratio is 1.5:1.

[0021] Preferably, the methacrylated hyaluronic acid is prepared by the following steps:

[0022] The hyaluronic acid is dissolved in deionized water, mechanically stirred until completely dissolved, and then methyl acrylate is added. The pH is adjusted to 8.5 with a base, and the reaction solution is stirred at room temperature. The reaction solution is then dialyzed and freeze-dried to obtain the methacrylated hyaluronic acid.

[0023] More preferably, the mass ratio of hyaluronic acid to methyl acrylate is 1:(1-3), and most preferably 1:2.6.

[0024] Preferably, the amount of HUCMSC cells added is 1 mL of culture medium containing 1 x 10 8 HUCMSC cells per 5 mL of hydrogel.

[0025] In a second aspect, the present application provides a self-oxygen-producing biological 3D printed mesenchymal stem cell liver organoid scaffold prepared according to the method.

[0026] In a third aspect, the present application also provides the use of the self-oxygen-producing biological 3D printed mesenchymal stem cell liver organoid scaffold in the preparation of a product for promoting the differentiation of functional stem cells.

[0027] In a fourth aspect, the present application also provides a CaO2@ZIF-8@SL nanoparticle, which is prepared by the following steps: CaO2 and 2-methylimidazole are added to water, mixed with Zn(CH3COO)2 and sodium lignosulfonate, incubated overnight, the solid is harvested and washed with deionized water, then dried to obtain the CaO2@ZIF-8@SL nanoparticle.

[0028] 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 most preferably the weight ratio is 32.3:3200:81:10. Preferably, the sodium lignosulfonate is in the form of a 2-5% volume concentration of sodium lignosulfonate aqueous solution.

[0029] In a fifth aspect, the present application also provides the use of the CaO2@ZIF-8@SL nanoparticle in promoting cell proliferation.

[0030] Preferably, the cells are human umbilical cord mesenchymal stem cells (HUCMSCs) or hepatocarcinoma cells (HepaRGs), but are not limited thereto.

[0031] The self-oxygen-producing biological 3D printing mesenchymal stem cell liver organ scaffold prepared by the method has the stability of releasing oxygen for a long time, good ROS scavenging capacity, and no cytotoxicity, and can be used for constructing a human liver organ model. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 The structure and composition characterization of CaO2@ZIF-8@SL are shown. (A) XRD of CaO2, ZIF-8 and CaO2@ZIF-8@SL; (B) Fourier infrared spectra of CaO2, SL, ZIF-8@SL, CaO2@ZIF-8@SL; (C) dissolved oxygen content; (D) particle size distribution of ZIF-8; (E) particle size distribution of CaO2@ZIF-8@SL; (F) DPPH free radical scavenging capacity; (G) SEM images of ZIF-8 and CaO2@ZIF-8@SL; (H) mapping images of CaO2@ZIF-8@SL; (I) EDS analysis of CaO2@ZIF-8@SL.

[0033] Figure 2 The physicochemical property characterization of the hydrogel scaffold material is 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; (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) Compression strength (strain of 30%) of HAMA / GelMA hydrogel; (I) Compression image of HAMA / GelMA hydrogel.

[0034] Figure 3 The cytotoxicity results of CaO2@ZIF-8@SL are shown. (A) Cytotoxicity of CaO2@ZIF-8@SL on HUCMSC cells; (B) Cytotoxicity of CaO2@ZIF-8@SL on HepaRG cells.

[0035] Figure 4 The cytotoxicity results of 3D printed HUCMSC-containing hydrogel are shown.

[0036] Figure 5 The live and dead cell staining results are shown.

[0037] Figure 6HUVEC cell tube formation results are shown. (A) Effect of 3D printed scaffolds in each group on HUVEC cell tube formation; (B) HUVEC cell tube formation indicators Nb branches, Nb junctions, and Tot. branching length.

[0038] Figure 7 In vitro stem cell induction differentiation QPCR detection results are shown. (A-H) 3D printed scaffold HUCMSCs cell differentiation ALB, AFP, CK18, HNF-1a, FOXA2, PRKACA, PRKACB, and PRKX gene qPCR detection results. DETAILED DESCRIPTION

[0039] The technical solutions of the present application are further described below in combination with the drawings and specific examples.

[0040] Unless otherwise specified, the instruments or reagents used in the examples of the present application are conventional instruments or reagents in the art, which are conventional products that can be purchased on the market. Unless otherwise specified, the specific experimental operations involved in the present application are understood or known by those skilled in the art according to their mastery of common knowledge or conventional technical means, and will not be described here.

[0041] 1 Preparation Example

[0042] 1.1 Preparation of CaO2 nanoparticles

[0043] 3g of calcium chloride was dissolved in 30mL of distilled water, 15mL of ammonia water (30wt%) was added to a 100mL beaker, stirred uniformly to obtain a calcium chloride-ammonia water mixed solution. 120mL of PEG200 was added to the calcium chloride-ammonia water mixed solution, ultrasonic for 15min, and stirred vigorously for 30min. 15mL of hydrogen peroxide was added dropwise at a speed of 10s / drop by a micro-injection pump. After 2 hours of reaction, 1M sodium hydroxide solution was slowly added dropwise in the suspension to pH 11.5, and after standing, the supernatant was discarded and the precipitate was retained. 0.1M sodium hydroxide solution was added to wash the precipitate, and after standing again, the supernatant was discarded and the precipitate was retained, which was repeated three times. The precipitate was collected by centrifugation (2000r / min) and freeze-dried to obtain yellow solid particles, which were stored in a vacuum drying oven at room temperature.

[0044] 1.2 Synthesis of ZIF-8 nanoparticles

[0045] 8.87g of 2-methylimidazole was dissolved in 50mL of deionized water, 500mg of zinc acetate hexahydrate was added, stirred at room temperature for 24 hours, centrifuged at 6000r / min for 10min, washed with deionized water for 3 times, and freeze-dried for 48 hours to obtain ZIF-8 nanoparticles.

[0046] 1.3 Preparation of CaO2@ZIF-8@SL nanoparticles

[0047] CaO2(32.3 mg) and 2-methylimidazole (3.2 g) were added to 13 mL of water, Zn(CH3COO)2(81 mg) and SL (sodium lignosulfonate) (10 mg SL) were added to 400 mL of water, after mixing all together, incubate at room temperature overnight. Harvest the solid and wash with deionized water, then dry the yellow solid at room temperature to obtain CaO2@ZIF-8@SL nanoparticles.

[0048] 1.4 Preparation of methacrylated gelatin (GelMA)

[0049] Weigh 5.0 g of gelatin and add it to 50 mL of PBS buffer, dissolve it completely in a 50 °C water bath with magnetic stirring to obtain a gelatin solution. Then slowly add 3 mL of methacrylic anhydride to the gelatin solution, stir at 700 rpm for 1 hour, maintain the reaction temperature at 50 °C, and the reaction solution contains a large number of oil droplets. After the reaction is completed, transfer the reaction solution to a cellulose dialysis bag with a molecular weight cutoff of 3500 Da, dialyze in deionized water at 40 °C for 3 days to remove byproducts. Collect the dialyzed reaction solution, centrifuge at 5000 rpm for 10 minutes to remove the precipitate, collect the supernatant, and freeze-dry at -80 °C to obtain the final product GelMA.

[0050] 1.5 Preparation of methacrylated hyaluronic acid (HAMA)

[0051] Weigh 5.0 g of hyaluronic acid and dissolve it in 400 mL of deionized water, mechanically stir until completely dissolved. Add 12 mL of methacrylic anhydride, adjust the pH of the reaction solution to 8.5 with 5M NaOH aqueous solution, stir at room temperature for 24 hours, then dialyze the reaction with a cellulose dialysis bag with a molecular weight cutoff of about 1.2 kDa, and freeze-dry to obtain HAMA.

[0052] 1.6 3D printing of GelMA / HAMA loaded with CaO2@ZIF-8@SL nanoparticles

[0053] Dissolve methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HAMA) in deionized water containing a certain proportion of LAP (light initiator, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone) and lemon yellow (light blocker, which helps to print small pore size) to prepare a bio-ink of GelMA / HAMA.

[0054] Specifically, first, the photo-initiation solution was prepared by adding 1 mg of LAP and 0.6 mg of lemon yellow into 1 mL of deionized water, vortexing for 30 s, and mixing uniformly to obtain the photo-initiation solution. 0.01 g of HAMA was dissolved in deionized water to obtain a 1% w / v HAMA solution, and a certain amount (0, 0.025 (2.5%), 0.050 (5%), 0.075 g (7.5%)) of GelMA was dissolved in the HAMA solution to obtain a GelMA / HAMA mixed solution, followed by the addition of 5 mg of CaO2@ZIF-8@SL.

[0055] Then, the prepared bio-ink was poured into the printer, the printing model and model size were selected, and the printing was formed (light intensity: 18 mW / cm 2 ; exposure time: 24 s; base layer number: 1; base layer exposure time: 26 s). Finally, the printed hydrogel was washed 2-3 times with sterile PBS to obtain the hydrogel scaffold.

[0056] 1.7 3D printing of GelMA / HAMA / HUCMSC loaded with CaO2@ZIF-8@SL nanoparticles

[0057] First, the photo-initiation solution was prepared by adding 1 mg of LAP and 0.6 mg of lemon yellow into 1 mL of deionized water, vortexing for 30 s, and mixing uniformly to obtain the photo-initiation solution. 0.01 g of HAMA was dissolved in deionized water to obtain a 1% w / v HAMA solution, and 0.075 g of GelMA was dissolved in the HAMA solution to obtain a GelMA / HAMA mixed solution, followed by the addition of 5 mg of CaO2@ZIF-8@SL to obtain a hydrogel solution. 5 mL of the hydrogel solution was taken, 1 mL of a-MEM complete medium containing 1 x 10 8 HUCMSC cells were added to obtain a bio-ink.

[0058] Then, the prepared bio-ink was poured into the printer, the printing model and model size were selected, and the printing was formed (light intensity: 18 mW / cm 2 ; exposure time: 24 s; base layer number: 1; base layer exposure time: 26 s). Finally, the printed hydrogel was washed 2-3 times with sterile PBS to obtain the hydrogel scaffold.

[0059] 2 Structural characterization and efficacy

[0060] 2.1 XRD

[0061] After the dried β-TCP was ground in a agate mortar, it was transferred to a test dish, and the structure of the material was tested by X-ray polycrystalline powder diffraction (XRD). The instrument used a copper (Cu) target K-ray, Voltage 40kV, current 40mA, scattering angle (2θ) is 10° to 80°.

[0062] In the CaO2 sample, the distinct characteristic diffraction peaks observed at 30.0° (002), 35.5° (110), 47.5° (112), and 52.5° (103) are characteristic of CaO2. Figure 1 A) in the diagram matches the ICDD card for CaO2 crystals. The synthesized ZIF-8 exhibits a highly crystalline structure, and its diffraction peaks agree well with the standard simulated XRD pattern of ZIF-8. However, some characteristic peaks of ZIF-8 are clearly visible in the XRD pattern of CaO2@ZIF-8@SL, which is due to the high crystallinity of the ZIF-8 in situ attached to CaO2. Furthermore, since ZIF-8 is grown in situ on its surface, there are no obvious characteristic peaks of CaO2 in the XRD pattern of CaO2@ZIF-8@SL.

[0063] 2.2 Infrared Detection

[0064] Weigh 8 mg of the sample to be tested and an appropriate amount of dry potassium bromide powder (mass ratio 5%) into an agate mortar, grind thoroughly to ensure uniform mixing, then take an appropriate amount of the ground sample powder and compress it into a tablet (vacuum pressure 20 mmHg, compression for 5 min) to obtain a sample thin film. Set the scanning range to 4000-400 cm⁻¹. -1 The detection was performed using a Fourier transform infrared spectrometer.

[0065] like Figure 1 As shown in Figure B, in the FTIR spectrum of CaO2, 873 cm⁻¹ -1 The absorption peak at [cm] is due to the O-O stretching vibration, which is the most important characteristic peak in CaO2, indicating the successful synthesis of CaO2. Furthermore, the asymmetric and symmetric stretching vibration absorption bands of the O-Ca-O group of CaO2 appear at 1419 and 1483 cm⁻¹, respectively. -1 Location. Among them, 1594cm -1 The absorption band at that location is attributed to the CO3 of calcium carbonate. 2- The carbonyl stretching vibration absorption peak indicates that the byproduct calcium carbonate is generated by the reaction of CaO2 with carbon dioxide during preparation and storage.

[0066] For ZIF-8@SL samples, 3134 and 2925 cm⁻¹ -1 The absorption bands at these locations represent the stretching of the aromatic and aliphatic CH groups of the imidazole ring, respectively, at 687-758 and 948-998 cm⁻¹. -1 The absorption bands at 3425 cm⁻¹ are associated with both in-plane and out-of-plane bending of the imidazole ring. Besides the characteristic bands of unmodified ZIF-8, there are also absorption bands at 3425 cm⁻¹. -1The broad absorption band in the vicinity is related to the -OH vibration in the SL structure and is located at 1424 cm⁻¹. -1 The absorption bands are linked to C-C bonds present in the SL and ZIF-8 structures, indicating that SL and ZIF-8 are bonded by hydrogen bonds. 2+ (Positive charge) tends to attach to the sulfonic acid group of SL.

[0067] In the FTIR of CaO2@ZIF-8@SL, tensile vibration peaks of OO and imidazole rings were observed, indicating that ZIF-8@SL was successfully attached in situ to the CaO2 surface.

[0068] 2.3 Determination of dissolved oxygen content

[0069] Add 1 mL of 30% H2O2 to 9 mL of pure water to obtain 3% H2O2. Calibrate the electrodes of the portable dissolved oxygen meter with 5% sodium sulfite. Then, add 1 mL of H2O2 to 100 mL of H2O, followed by 2 mg of CaO2@ZIF-8@SL. Measure the dissolved oxygen concentration in the H2O group, the H2O+H2O2 group, and the H2O+H2O2+CaO2@ZIF-8@SL group, respectively.

[0070] like Figure 1 As shown in C, since there is no peroxide or oxygen source in pure water, the dissolved oxygen remains near the saturation value of the natural environment and does not increase or decrease significantly. Hydrogen peroxide slowly decomposes in water to generate oxygen (2H2O2→2H2O+O2), thus increasing the dissolved oxygen content in this group. Due to the dual effects of CaO2@ZIF-8@SL and hydrogen peroxide, the CaO2@ZIF-8@SL group has the highest dissolved oxygen level. In addition, due to the encapsulation effect of ZIF-8@SL, the CaO2@ZIF-8@SL group exhibits stability in oxygen release over a longer period of time.

[0071] 2.4DLS

[0072] Nanoparticles were dispersed in deionized water to ensure uniform dispersion, and their particle size and potential were measured using a laser nanoparticle size analyzer.

[0073] Particle size analysis of ZIF-8 nanoparticles and CaO2@ZIF-8@SL nanoparticles was performed using a laser nanoparticle size analyzer. Figure 1 As shown in Figures D and E, the average particle size of ZIF-8 is 412.8 ± 14.08 nm, while the average particle size of CaO2@ZIF-8@SL is 484.3 ± 38.52 nm. This is likely because the internal loading of CaO2 into ZIF-8 increases the particle size. Secondly, the SL layer, which coats the outside of the particles, directly increases the particle size. Additionally, agglomeration may occur during the synthesis process, leading to particle aggregation and thus an increase in particle size.

[0074] 2.5 ROS removal capability test

[0075] The antioxidant properties of microgel assemblies were evaluated using a method for scavenging 1,1-diphenyl-2-pyridylhydrazide (DPPH) free radicals. Microgel samples with different components were incubated with a mixture of DPPH reagent in the dark for 30 minutes, and the remaining DPPH was analyzed using UV-Vis spectroscopy. The formula for determining the DPPH scavenging rate is as follows:

[0076] D(%) = [[A blank - (A assay - A control)] / A blank] × 100%

[0077] like Figure 1 As shown in F, the H2O group and the H2O+H2O2 group showed no obvious free radical scavenging activity, while the H2O+H2O2+CaO2@ZIF-8@SL group showed a significant lightening of the purple color. This may be because sodium lignosulfonate contains multiple phenolic hydroxyl groups (-OH), which can interact with DPPH radicals (2,2-diphenyl-1-picrylhydrazine radicals) through hydrogen atom transfer reaction (HAT), reducing them to yellow diphenyl-pyridinemethylhydrazine. In addition, the aromatic ring in sodium lignosulfonate has a π-electron system, which can participate in free radical scavenging reactions, donating electrons to free radicals through electron transfer reaction (ETr), thereby converting free radicals into stable non-free radical forms.

[0078] 2.6SEM-EDS

[0079] The prepared material was attached to conductive adhesive, and gold was sprayed 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.

[0080] Figure 1 Figure G shows SEM images of two types of nanoparticles (ZIF-8 and CaO2@ZIF-8@SL). As can be seen from the image, the unmodified ZIF-8 nanoparticles have a uniform rhombic dodecahedral morphology, a smooth surface, and an average particle size of approximately 100 nm. However, after loading with CaO2 and SL coatings, the CaO2@ZIF-8@SL surface becomes rough, and the average particle size decreases to approximately 70 nm, showing a more restricted particle size distribution. This indicates that CaO2 and SL, acting as growth inhibitors, can modulate the nucleation rate of ZIF-8 and limit its growth, thereby effectively controlling the size of ZIF-8 crystals during synthesis.

[0081] Characterized by EDS ( Figure 1 The elemental composition of ZIF-8@SL nanoparticles was analyzed, and the presence of the required elements (Zn, Ca, S, O) in the structure was confirmed.

[0082] 3. Structural composition and physicochemical characterization of hydrogels

[0083] 3.1 Infrared

[0084] Weigh 8 mg of the sample to be tested and an appropriate amount of dry potassium bromide powder (approximately 5% by mass) into an agate mortar. Grind thoroughly to ensure uniform mixing. Take an appropriate amount of the ground sample powder and compress it into a tablet (vacuum pressure 20 mmHg, compression time 5 min) to obtain a sample thin film. Set the scanning range to 4000-400 cm⁻¹. -1 The detection was performed using a Fourier transform infrared spectrometer.

[0085] 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.

[0086] 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.

[0087] 3.2 Nuclear Magnetic Resonance Imaging

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 3.3 Rheology test

[0092] Rheology measurements were performed using a stainless steel parallel plate geometry 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 sweep was performed at room temperature from 0.1 to 100 rad / s to determine the linear viscoelastic region of the hydrogel, and the storage modulus (G') and loss modulus (G") curves were recorded.

[0093] Figure 2 E and F in FIG. 3E and FIG. 3F respectively show the relationship between the storage modulus (G') and the loss modulus (G") of the hydrogel and the frequency and time. When G' is greater than G", the hydrogel exhibits a gel state. As shown in the figures, after the hydrogel is gelled, G' is always greater than G" with the increase of time and angular frequency, which indicates that the hydrogel forms a stable three-dimensional network structure, thereby maintaining its gel state. Moreover, with the increase of the concentration of GelMA, G' increases continuously, which indicates that the elastic properties of the hydrogel scaffold gradually increase with the increase of the concentration of GelMA, indicating that the addition of GelMA can effectively improve the elastic properties of the hydrogel scaffold. This may be because: with the increase of the concentration of GelMA, the crosslinking density also increases, more crosslinking points are formed between GelMA chains, resulting in a stronger network structure. Secondly, with the increase of the concentration of GelMA, the intermolecular force also increases, making the crosslinked network of the hydrogel more compact, and improving the elastic properties.

[0094] 3.4 Compression modulus

[0095] 500 μL of the hydrogel was prepared into a highly uniform cylinder with a bottom diameter of 11 mm. Then the prepared hydrogel sample was placed on the sample table of the universal testing machine, and the height of the clamp was adjusted so that the upper and lower bottom surfaces of the hydrogel were in contact with the clamp. Then the compression rate was set to 0.05 mm / s, and the sample was compressed at a constant rate to 70% of the strain, and the pressure and displacement values were recorded. The compression modulus was calculated according to the following formula:

[0096]

[0097] where F is the recorded pressure value, and S is the cross-sectional area of the hydrogel sample.

[0098] Figure 2G, H and I in the table are the stress-strain curve, compression strength at 30% deformation and compression image of the hydrogel, respectively. The hydrogel of 1% H ruptured easily and could not recover to the original shape by light pressing. The hydrogel with 7.5% GelMA could resist certain pressure and recover to the original shape after pressing, which proved that the mechanical properties of the hydrogel could be improved by adding GelMA. As can be seen from the stress-strain curve, the compression strength of the hydrogel increased with the increase of the concentration of GelMA. The compression strength of 1% H was 0.37±0.07 kPa, the compression strength of 1% HAMA / 2.5% GelMA was 4.47±0.10 kPa, and the compression strength of 1% HAMA / 5% GelMA was 9.22±0.09 kPa. The compression strength of 1% HAMA / 7.5% GelMA was 12.55±0.86 kPa. This may be due to the fact that the higher the concentration of GelMA, the more compact the cross-linked network formed, and the greater the intermolecular force, thereby producing stronger resistance to external force and stronger mechanical properties. The modulus of the hydrogel matching the elastic properties of the liver is about 1-10 kPa. In combination with the rheological properties, 1% HAMA / 5% GelMA and 1% HAMA / 7.5% GelMA can be selected as the combination of the hydrogel for treating liver damage, which is more conducive to the recovery of liver damage.

[0099] Cytotoxicity of CaO2@ZIF-8@SL

[0100] After the CaO2@ZIF-8@SL powder was irradiated with ultraviolet light overnight, a CaO2@ZIF-8@SL solution with a concentration of 10 mg / mL was prepared with sterile PBS, and diluted to 5, 2.5, 0.5, 0.25, 0.1, 0.05, 0.025 mg / mL with DMEM complete medium.

[0101] HUCMSC cells and liver cells HepaRG cells were digested with 0.05% trypsin, and the HUCMSC cells were resuspended in α-MEM complete medium, and the liver cells HepaRG were resuspended in DMEM complete medium, and seeded in a 96-well plate at a density of 5000 / cell per well, and cultured in a 37°C constant temperature carbon dioxide incubator (containing 5% CO2) for 24 h to allow the cells to adhere. The culture medium was aspirated and washed with PBS, and different concentrations (5, 2.5, 0.5, 0.25, 0.1, 0.05, 0.025 mg / mL) of CaO2@ZIF-8@SL solution were added for continuous culture for 24 h. After the culture was completed, the culture medium was aspirated and washed with PBS 3 times, 100 μL of CCK-8 working solution was added to each well, and incubated in a 37°C constant temperature carbon dioxide incubator (containing 5% CO2) for 30 min. Then the absorbance (OD) was measured at 450 nm wavelength by a microplate reader, and the cell survival rate was calculated according to the following formula:

[0102]

[0103] The effects of different concentrations of CaO2@ZIF-8@SL solution on the viability of HUCMSCs were evaluated using the CCK-8 assay. Figure 3 As shown in Figure A, the activity of HUCMSCs exhibited a certain trend with increasing drug concentration (0.025 mg / mL-5 mg / mL). The activity of HUCMSCs reached its highest level of 112.92% at a drug concentration of 0.05 mg / mL, while the activity was 94.37% at 5 mg / mL. This indicates that excessively high concentrations of CaO2@ZIF-8@SL solution have toxic side effects on HUCMSCs, while drug concentrations of 0.025 mg / mL-0.1 mg / mL promote HUCMSC cell proliferation.

[0104] The effects of different concentrations of CaO2@ZIF-8@SL solution on the viability of HepaRG cells were evaluated using the CCK-8 assay. Figure 3 As shown in Figure B, the activity of HepaRG cells also showed a certain trend with increasing drug concentration (0.025 mg / mL-5 mg / mL). The activity of HepaRG cells reached its highest level of 111.54% at a drug concentration of 0.05 mg / mL, while it was 91.78% at 2.5 mg / mL. This indicates that excessively high concentrations of CaO2@ZIF-8@SL solution have toxic side effects on HepaRG cells, while drug concentrations between 0.025 mg / mL and 0.25 mg / mL promote the proliferation of HepaRG cells.

[0105] 5. Cytotoxicity of 3D-printed hydrogels containing HUCMSCs

[0106] HUCMSC cells were digested with 0.05% trypsin, and 1×10⁻⁶ cells were added. 8HUCMSC cells were resuspended in 1 mL of a-MEM complete medium, and the a-MEM complete medium containing the cells was mixed with 5 mL of each group of hydrogel (GelMA / HAMA, GelMA / HAMA / CaO2@ZIF-8@SL) solution, respectively, and printed into 8x8x1.5 mm hydrogel scaffolds (GHH, GHHC) using a 3D printer, and cultured with a-MEM complete medium. They were placed in a 48-well plate and cultured for 24, 72, and 120 h, respectively. After the culture was completed, the medium was aspirated and washed with PBS three times, 100 μL of CCK-8 working solution (containing 10% CCK-8 in a-MEM basic medium) was added to each well, and incubated in a 37°C constant temperature carbon dioxide incubator (containing 5% CO2) for 30 min. Then the hydrogel scaffolds were broken and centrifuged, and the supernatant of each group was transferred to a 96-well plate. The absorbance (O D) was measured at 450 nm wavelength using a microplate reader, and the cell survival rate was calculated according to the following formula:

[0107]

[0108] The CCK-8 method was used to evaluate the effect of each group of materials on the proliferation activity of HUCMSC cells. As shown in Figure 4 , the survival rate of HUCMSC cells gradually increased as the culture time was prolonged (Day 1, Day 3, Day 5), indicating that the materials had no significant cytotoxicity. Notably, the GHHC group showed a significant proliferative effect: on the third day, its cell activity was significantly higher than that of the GHH group (p<0.05), and by the fifth day, the HUCMSC cell activity of the GHHC group was significantly improved, indicating that it had a better effect on cell growth.

[0109] 6 Live and dead cell staining

[0110] HUCMSC cells were digested with 0.05% trypsin, and 1x10 8 HUCMSC cells were resuspended in 1 mL of a-MEM complete medium, and the a-MEM complete medium containing the cells was mixed with 5 mL of each group of hydrogel (GelMA / HAMA, GelMA / HAMA / CaO2@ZIF-8@SL) solution, respectively, and printed into 8x8x1.5 mm hydrogel scaffolds (GHH, GHHC) using a 3D printer, and cultured in a 37°C constant temperature carbon dioxide incubator (containing 5% CO2) for 24, 72, and 120 h. The medium was aspirated and washed with PBS three times, and live and dead staining working solution (2 μM calcein AM, 8 μM PI) was added and incubated at room temperature for 20 min. After washing with PBS three times, the pictures were immediately captured using a fluorescence microscope, and the live and dead cells were analyzed. Calcein AM labeled live cells emitted green fluorescence, and propidium iodide (PI) labeled dead cells emitted red fluorescence.

[0111] Figure 5 The effect of different 3D printed hydrogel scaffolds on HUCMSCs survival was evaluated by live-dead cell staining. Compared with the control group, no obvious PI positive signal (dead cells) was observed in each experimental group, indicating that the materials had good biocompatibility and no significant cytotoxicity. The Calcein-AM positive signal (live cells) of the GHHC group was significantly enhanced, with denser cell distribution and better proliferation status.

[0112] 7 In vitro pro-angiogenesis

[0113] First, Matrigel after thawing was plated onto a 48-well plate at a volume of 100 μL / well and incubated in a cell culture incubator for 30 min to convert from a liquid state to a solid gel. HUVECs were seeded onto the Matrigel surface at a cell density of 1 x 10 5 / well, and a Transwell chamber was added, with cells in the lower chamber and 3D printed scaffolds in the upper chamber of the Transwell. α-MEM complete medium was added. After 6 h of incubation at 37°C, the tubular network structure formed by the cells was observed under a microscope, and ImageJ software was used to calculate the tubular network structure parameters, including the number of nodes, the number of grids, and the number of trunks.

[0114] Figure 6 A in FIG. 7 is a fluorescence image of HUVEC cell tube formation, in which human umbilical cord endothelial cells HUVECs treated with GHHC form a denser tubular network. Figure 6 B in FIG. 7 is a tube formation index statistical chart, and the total branch length and node number of HUVECs after GHHC treatment have a significant difference (p < 0.001) compared with the control group, indicating that the GHHC material can effectively promote angiogenesis.

[0115] 8 RT-qPCR detection of the expression level of specific genes for stem cell induced differentiation in vitro

[0116] To induce liver-derived differentiation, the 3rd passage of HUCMSC cells was printed with bio-ink according to the above grouping, and then placed 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) for 2 weeks. Subsequently, the medium was changed to DMEM / F-12 medium containing 0.5 μmol / L dexamethasone, 20 ng / ml HGF, 10 ng / ml Oncostatin M (Peprotech), and 1% ITS, and cultured for 2 weeks.

[0117] Cells were collected and the expression of hepatocyte line markers (AFP, CK-18, ALB, Hnf1a, Foxa2, Prkaca, Prkacb, Prkx and GAPDH) was detected by real-time RT-qPCR. The cycling conditions were: 95°C for 10 min, followed by 40 cycles of 95°C for 10 s, 60°C for 20 s and 72°C for 15 s.

[0118] The primers used were designed as follows (forward and reverse):

[0119] ALB (albumin):

[0120] 5'-AGAGGTCTCAAGAAACCTAGGAAA-3'; 5'-GGTTCAGGACCACGGATAGA-3'.

[0121] AFP:

[0122] 5'-TGCAAACGATGAAGCAAGAG-3'; 5'-AACAGGCCTGAGAAATCTGC-3'.

[0123] CK18:

[0124] 5'-TGATGACACCAATATCACACGA-3'; 5'-CTGGGCTTGTAGGCCTTTTA-3'.

[0125] Hnf1a: 5'-GACATGCTTCAGTGGGAGAA-3' and 5'-GAGCTGTCTGAGGAGCTGTG-3'.

[0126] Foxa2: 5'-TCGGAGGAGGAGGAGGAGAG-3' and 5'-CGGAGGAGGGAGGAGGAGAG-3'.

[0127] Prkaca: 5'-ATGGAGGACAGGAGAGGAGG-3' and 5'-AGGAGGGAGGAGGAGGAGG-3'

[0128] Prkacb: 5'-ATGAGGAAGAGGAGGAGGA-3' and 5'-AGGAGGAGGAGGAGGAAGG-3'

[0129] Prkx: 5'-ATGGAGGAGGAGGAGGAAGG-3' and 5'-AGGAGGAGGAGGAGGAGGA-3'.

[0130] GAPDH (glyceraldehyde 3-phosphate dehydrogenase):

[0131] 5'-ACACCCACTCCTCCACCTTT-3'; 5'-TTACTCCTTGGAGGCCATGT-3'.

[0132] Detailed steps are as follows:

[0133] (1) Extraction of total RNA

[0134] 1) Sample processing: centrifugal collection of cells into a 1.5 mL centrifuge tube, addition of 1 mL of RNAiso Plus, shaking and mixing, and then room temperature standing for about 5 min.

[0135] 2) Phase separation: addition of 0.2 mL of chloroform per 1 mL of RNAiso Plus, shaking and mixing for 15 s, and then room temperature standing for about 3 min, 4°C, 12000 rpm, centrifugation for 10 min.

[0136] 3) Precipitation: transfer of the aqueous phase to a new 1.5 mL centrifuge tube, addition of 0.5 mL of isopropanol per 1 mL of RNAiso Plus, mixing, and then room temperature standing for 10 min, 4°C, 12000 rpm, centrifugation for 10 min.

[0137] 4) Washing: discarding the supernatant, addition of 1 mL of 75% ethanol per 1 mL of RNAiso Plus, mixing, and then 4°C, 7500 rpm, centrifugation for 5 min.

[0138] 5) Dissolution: discarding the supernatant, air-drying of the RNA precipitate for about 5 min (note that it should not be completely dried, only the precipitate should be whitish), and then addition of an appropriate amount of DEPC-treated water to dissolve the RNA precipitate.

[0139] 6) Determination of concentration and purity: determination of the RNA concentration and purity using a spectrophotometer, and then recording the data.

[0140] (2) First-strand cDNA synthesis:

[0141] 1) The reverse transcription reaction solution is prepared according to the following component allocation, and the preparation of the reaction solution should be completed on ice.

[0142]

[0143] 2) The reaction is carried out according to the following procedure: 25°C, 5 min; 42°C, 30 min; 85°C, 5 min.

[0144] 3) After the reaction is completed, the product is diluted 10 times and stored at -20°C for standby use.

[0145] (3) Quantitative PCR detection

[0146] 1) Dissolve the Mix at 4°C, gently invert and mix, and then centrifuge briefly.

[0147] 2) Prepare the reaction solutions shown in the table below on ice.

[0148] Ingredient Amount Final Concentration SYBR Green Master Mix 5 μL 1 x Mix Forward Primer (2 μM) 1 μL 0.2 μM Reverse Primer (2 μM) 1 μL 0.2 μM cDNA 3 μL RNase-free Water to 10 μL

[0149] 3) Briefly centrifuge the reaction tube to ensure that all reaction liquid is at the bottom of the reaction well.

[0150] 4) The reaction is carried out using the following procedure.

[0151]

[0152] qPCR analysis showed that the GHHC group had a more significant promoting effect on the differentiation of HUCMSCs into hepatocytes. Compared with the GHH group, the GHHC group significantly upregulated the expression of mature hepatocyte markers ALB, AFP, and CK18. Figure 7 The presence of AC in the data indicates that it can effectively promote the functional maturation of hepatocytes. The expression of key regulatory factors for hepatocyte development, HNF-1α and FOXA2, was also significantly enhanced. Figure 7 (D and E in the original text). Furthermore, the expression of PRKACA, PRKACB, and PRKX genes, which are closely related to hepatocyte metabolism, was significantly upregulated. Figure 7 Further studies using FH (Fluorescent HCG) confirmed that GHHC can promote the functional maturation of hepatocytes. The results showed that GHHC significantly enhanced the ability of HUCMSCs to differentiate into functional hepatocytes by regulating key hepatocyte differentiation factors and metabolism-related genes.

[0153] 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 self-oxygen-producing biological 3D printed mesenchymal stem cell liver organoid scaffold, characterized in that, The method comprises the following steps: S1, preparing CaO2@ZIF-8@SL nanoparticles; S2, adding CaO2@ZIF-8@SL nanoparticles into a mixed solution of methacrylated gelatin and methacrylated hyaluronic acid to obtain a hydrogel solution; S3, adding HUCMSC cells into the hydrogel solution to obtain a bio-ink, and performing 3D printing to obtain the self-oxygen-producing biological 3D printed mesenchymal stem cell liver organoid scaffold.

2. The method of claim 1, wherein, The step S1 comprises: adding CaO2 and 2-methylimidazole into water, mixing with Zn(CH3COO)2 and sodium lignosulfonate, incubating overnight, harvesting the solid and washing with deionized water, and then drying to obtain CaO2@ZIF-8@SL nanoparticles.

3. The method of claim 2, wherein, The weight ratio of CaO2, 2-methylimidazole, Zn(CH3COO)2 and sodium lignosulfonate is (30-35):(3000-3500):(75-85):(8-12).

4. The method of claim 1, wherein, The step S2 comprises: The methacrylated gelatin and the methacrylated hyaluronic acid are dissolved in deionized water containing LAP and tartrazine, uniformly mixed, then the CaO2@ZIF-8@SL nanoparticles are added and uniformly mixed, and then the prepared bio-ink is printed and washed to obtain the self-oxygen-producing biological 3D printed mesenchymal stem cell liver organoid scaffold.

5. The method of claim 4, wherein, The methacrylated gelatin is prepared by the following steps: Methyl acrylate is added dropwise into PBS buffer containing gelatin, magnetically stirred, and after the reaction is completed, the by-products are removed by dialysis in deionized water, the reaction solution after dialysis is collected, the precipitate is removed by centrifugation, the supernatant is collected, and freeze-drying is performed to obtain the final product, methacrylated gelatin.

6. The method of claim 5, wherein, The mass ratio of gelatin to methyl acrylate is (1-2):

1.

7. The method of claim 4, wherein, The methacrylated hyaluronic acid is prepared by the following steps: Hyaluronic acid is dissolved in deionized water, mechanically stirred until completely dissolved, methyl acrylate is added, the pH is adjusted to 8.5 with a base, stirred at room temperature, and then the reaction solution is dialyzed and freeze-dried to obtain methacrylated hyaluronic acid.

8. The method of claim 7, wherein, The mass ratio of hyaluronic acid to methyl acrylate is 1:(1-3).

9. The method according to any one of claims 1 to 8, characterized in that, In the step S2, the mass ratio of methacrylated gelatin, methacrylated hyaluronic acid and CaO2@ZIF-8@SL nanoparticles is (10-15):2:

1.

10. The method according to any one of claims 1 to 8, characterized in that, In the step S2, the HUCMSC cells are added in an amount of 1 mL of culture medium containing 1 x 10 8 HUCMSC cells per 5 mL of hydrogel.

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