Hydrogel capable of regulating stem cell differentiation as well as preparation method and application of hydrogel

Abstract

The invention relates to hydrogel capable of regulating stem cell differentiation as well as a preparation method and application thereof, and belongs to the technical field of biological tissue medical engineering. According to the preparation method disclosed by the invention, the dECM-ODex hydrogel of which the rigidity and viscoelasticity can be independently adjusted is synthesized by utilizing an acellular matrix (dECM) and oxidized dextran (ODex). The dECM contains a large amount of collagen, the collagen forms a main gel network skeleton during polymerization, a weak Schiff base reaction exists between the dECM and ODex, and the mechanical properties, including viscoelasticity and rigidity, of the dECM-ODex hydrogel can be adjusted by keeping the concentration of the dECM hydrogel and adjusting the number of aldehyde groups (namely the molar ratio of NaIO4 to Dex and the concentration of ODex) of the ODex at the same time. And a liver tissue mechanical environment is simulated. The synthesized dECM-ODex hydrogel can be used for regulating and controlling the differentiation of stem cells and treating liver diseases. The invention provides a new tissue engineering material selection for the treatment of liver diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of biological tissue medical engineering, and in particular to a hydrogel capable of regulating stem cell differentiation, and a preparation method and application thereof. Background Art

[0002] Many genetic and metabolic diseases affect the liver, leading to fibrosis and cirrhosis. The only therapeutic intervention for end-stage liver disease is orthotopic liver transplantation. However, effective treatments, including liver and hepatocyte transplantation, face the problem of insufficient donors. Therefore, hepatocyte-like cells (HLCs) derived from human adipose-derived mesenchymal stem cells (hADSCs), which are abundant in source and have low immunogenicity, have great application prospects. However, simple cell transplantation for the treatment of liver disease has low efficiency in reaching the liver site, and most implanted cells are trapped in the lungs after intravenous injection or cleared by macrophages after spleen transplantation. In addition, excessive reactive oxygen species and inflammatory responses in damaged tissues can also impair the survival of transplanted cells, severely limiting the effectiveness of ALF treatment. In response to the above problems, biomaterial-loaded cells can be used for liver tissue engineering to improve cell survival in mouse models of liver disease. In this tissue engineering, biomaterials create a three-dimensional culture environment full of biochemical and biophysical signals for transplanted cells, promoting interactions between cells and extracellular matrix. The extracellular microenvironment triggers biochemical signals through mechanical transduction, enabling cells to sense, respond and adapt to their physical environment, thereby regulating cell proliferation, paracrine secretion, stem cell differentiation and other behaviors, affecting the fate of stem cells. Optimizing the mechanical properties of the extracellular microenvironment and accurately replicating the stem cell niche can improve the efficiency of liver organoid derivation and bridge the functional gap between human pluripotent stem cell-derived hepatocytes and human primary hepatocytes.

[0003] Engineered hydrogels in three-dimensional cell culture are often used to simulate the extracellular microenvironment and liver tissue engineering. The stiffness of a material is a mechanical property that has been widely shown to control different stem cell activities. Previous studies have found that hydrogel matrix stiffness has a regulatory effect on the differentiation of hepatocyte-like cells or liver organoids into primary hepatic progenitor cells, embryonic stem cells, and human pluripotent stem cells. In two-dimensional culture, matrix stiffness determines the fate of stem cells; in three-dimensional culture, the stiffness characteristics that simulate the natural liver niche are more conducive to improving the liver differentiation of stem cells and the function of hepatocyte-like cells. The viscoelastic behavior of hydrogels has been confirmed to have an impact on endothelial cell migration and angiogenesis, neural maturation and morphogenesis of human induced pluripotent stem cells, chondrogenesis of mesenchymal stem cells, and collective cell spatiotemporal organization.

[0004] The synthetic hydrogels in the field of stem cell and liver regenerative medicine currently studied have the problem of needing to add additional cell adhesion factors and create a biochemical environment, resulting in high costs; and in terms of the physical properties of the hydrogel, its stiffness and viscoelasticity are difficult to independently adjust and screen. Many human tissues, including the liver, are not purely elastic, but viscoelastic, and show time dependence on the applied force. Therefore, simply adjusting the stiffness of the hydrogel that simulates the extracellular matrix is ​​not enough to fully reflect the mechanical properties of the extracellular microenvironment during the hepatic differentiation of stem cells, and it is difficult to accurately regulate the hepatic differentiation of stem cells. The technical focus of existing hydrogel matrices is mostly on regulating the proliferation, migration, differentiation and other behaviors of stem cells, and rarely using the hydrogel-stem cell construct to go deep into the application of biomaterial-cell disease treatment. Therefore, further research and development of hydrogels is very necessary. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a hydrogel capable of regulating stem cell differentiation and a preparation method and application thereof.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a method for preparing a hydrogel capable of regulating stem cell differentiation, comprising the following steps:

[0008] S1. Mixing dried decellularized matrix (dECM) and protease and dissolving them in a hydrochloric acid solution to prepare a decellularized matrix solution; the mass ratio of the decellularized matrix to the protease is (5-20):1;

[0009] S2, mixing sodium periodate and dextran (Dex) and dissolving them in water, protecting from light for full reaction, and then adding ethylene glycol to terminate the reaction; dialysis purification to remove residual sodium periodate and other small molecules, and drying the purified solution to obtain oxidized dextran (ODex); the molar ratio of sodium periodate to dextran is (0.5-1.5):1;

[0010] S3. Dissolve oxidized dextran (ODex) in a solvent to obtain an oxidized dextran solution, and then mix it with the decellularized matrix solution of step S1, obtain a pregel through a Schiff base reaction, and incubate the pregel at 35 to 40°C to obtain the hydrogel (dECM-ODex hydrogel); the solvent includes water, PBS or cell culture medium; the mass ratio of the decellularized matrix to the oxidized dextran in the hydrogel is 1: (0.1 to 1).

[0011] The present invention utilizes the primary amine in the decellular matrix and the aldehyde group of the oxidized dextran to react with a Schiff base reaction (Schiffbase) to obtain the first layer network structure (pregel) of the dECM-ODex hydrogel, and then polymerizes the collagen inside the decellular matrix by a 37°C reaction to prepare a double-layer network of dECM-ODex hydrogel. The hydrogel prepared by the preparation method of the present invention is a viscoelastic layered porous structure, and the stiffness is within the stiffness range of natural liver tissue. The hydrogel prepared by the present invention is non-toxic to cells, has good biocompatibility, has biodegradability and can be suitable for long-term cell culture. As a culture matrix, the mechanical properties will not change, the stability is good, and the expression of genes and proteins related to stem cell differentiation can be promoted, and the differentiation of stem cells can be promoted. The hydrogel alone, the hydrogel encapsulating stem cells, and the hydrogel encapsulating stem cell differentiation cells can promote the migration of human umbilical vein endothelial cells (HUVECs), promote angiogenesis, and have the ability to remove ROS, promote the M2 polarization of macrophages, and promote the expression of anti-inflammatory genes. It can improve the morphology of the liver in acute liver failure and has the function of treating liver diseases such as acute liver failure.

[0012] As a preferred embodiment of the preparation method of the present invention, in step S1, the decellularized matrix (dECM) includes liver decellularized matrix, heart decellularized matrix or kidney decellularized matrix.

[0013] As a preferred embodiment of the preparation method of the present invention, the decellularized matrix (dECM) is a liver decellularized matrix.

[0014] As a preferred embodiment of the preparation method of the present invention, in step S1, the protease includes pepsin, trypsin, papain or collagenase; and the concentration of the hydrochloric acid solution is 0.1M to 1M.

[0015] As a preferred embodiment of the preparation method of the present invention, the protease is pepsin; and the concentration of the hydrochloric acid solution is 0.1M.

[0016] As a preferred embodiment of the preparation method of the present invention, in step S1, the mass ratio of the decellularized matrix to the protease is 10:1.

[0017] As a preferred embodiment of the preparation method of the present invention, in step S2, the reaction temperature of the sufficient reaction is 20°C to 30°C, and the reaction time is 3h to 5h. As a preferred embodiment of the preparation method of the present invention, the reaction temperature of the sufficient reaction is 25°C, and the reaction time is 4h.

[0018] As a preferred embodiment of the preparation method of the present invention, in step S2, the molar ratio of sodium periodate to dextran is 0.5:1.

[0019] As a preferred embodiment of the preparation method of the present invention, in step S3, before the mixing, the pH value of the decellularized matrix solution is adjusted to 6.5-7.5. As a preferred embodiment of the preparation method of the present invention, in step S3, before the mixing, the pH value of the decellularized matrix solution is adjusted to 7.

[0020] As a preferred embodiment of the preparation method of the present invention, in step S3, the cell culture medium includes DMEM basal culture medium, stem cell serum-free culture medium, DMEM / F12 cell culture medium, or RPMI-1640 cell culture medium.

[0021] As a preferred embodiment of the preparation method of the present invention, in step S3, the reaction temperature of the Schiff base reaction is 20° C. to 30° C., and the reaction time is 10 min to 60 min.

[0022] As a preferred embodiment of the preparation method of the present invention, in step S3, the incubation time is 30 min to 60 min. As a preferred embodiment of the preparation method of the present invention, in step S3, the incubation temperature is 37° C., and the incubation time is 30 min.

[0023] As a preferred embodiment of the preparation method of the present invention, in step S3, the mass ratio of the decellularized matrix to the oxidized dextran in the hydrogel is 1:(0.25-1). As a preferred embodiment of the preparation method of the present invention, in step S3, the mass ratio of the decellularized matrix to the oxidized dextran in the hydrogel is 1:0.25.

[0024] As a preferred embodiment of the preparation method of the present invention, in step S3, the concentration of the decellularized matrix (dECM) in the hydrogel is 1% w / v, and the concentration of oxidized dextran (ODex) is 0.25% w / v to 1% w / v. As a preferred embodiment of the preparation method of the present invention, in step S3, the concentration of the decellularized matrix (dECM) in the hydrogel is 1% w / v, and the concentration of oxidized dextran (ODex) is 0.25% w / v.

[0025] In a second aspect, the present invention provides a hydrogel prepared by the above preparation method.

[0026] In a third aspect, the present invention provides application of the above hydrogel in regulating stem cell differentiation.

[0027] In a fourth aspect, the present invention provides use of the above hydrogel in preparing a product for treating liver diseases.

[0028] The hydrogel of the invention can improve the symptoms of liver diseases such as acute liver failure, and can be prepared into a product for treating liver diseases.

[0029] As a preferred embodiment of the application of the present invention, the liver disease includes liver failure or liver fibrosis. As a preferred embodiment of the application of the present invention, the liver failure includes chronic liver failure or acute liver failure.

[0030] In a fifth aspect, the present invention provides the use of the above hydrogel in combination with stem cells in the preparation of a product for treating liver diseases. The hydrogel of the present invention is used to encapsulate stem cells, which can better promote the migration of human umbilical vein endothelial cells (HUVECs), angiogenesis, M2 polarization of macrophages, and expression of anti-inflammatory genes, has a higher ROS scavenging ability, and has a better therapeutic effect on liver diseases.

[0031] As a preferred embodiment of the application of the present invention, the stem cells include mesenchymal stem cells, embryonic stem cells or human induced pluripotent stem cells. As a preferred embodiment of the application of the present invention, the mesenchymal stem cells include human adipose-derived mesenchymal stem cells (hADSCs) or bone marrow mesenchymal stem cells.

[0032] In a sixth aspect, the present invention provides a product for treating liver diseases, comprising the hydrogel.

[0033] As a preferred embodiment of the product of the present invention, the product further comprises stem cells. As a preferred embodiment of the product of the present invention, the stem cells comprise mesenchymal stem cells, embryonic stem cells or human induced pluripotent stem cells. As a preferred embodiment of the product of the present invention, the mesenchymal stem cells comprise human adipose-derived mesenchymal stem cells (hADSCs) or bone marrow mesenchymal stem cells.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention utilizes decellularized matrix (dECM) and oxidized dextran (ODex) to synthesize dECM-ODex hydrogels that can independently adjust stiffness and viscoelasticity. dECM contains a large amount of collagen, which forms the main gel network skeleton when polymerized, and there is a weak Schiff base reaction between dECM and ODex. By maintaining the concentration of dECM hydrogel while adjusting the number of aldehyde groups of ODex (i.e., the molar ratio of the oxidant sodium periodate NaIO4 to dextran Dex and the ODex concentration), the mechanical properties of dECM-ODex hydrogels, including viscoelasticity and stiffness, can be adjusted to simulate the mechanical environment of liver tissue. The synthesized dECM-ODex hydrogel can regulate the differentiation of stem cells and treat liver diseases. The present invention provides a new tissue engineering material option for the treatment of liver diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1Schematic diagram of the adjustable biomechanical hydrogel regulating the hepatic differentiation of mesenchymal stem cells and treating acute liver failure in an embodiment of the present invention, wherein A represents the preparation of three hydrogels with different mechanical properties and their effects on the morphology of hADSCs, B represents the Faster Softer hydrogel promoting the hepatic differentiation of hADSCs and the paracrine activity of HLCs, and C represents the therapeutic effect of Faster SofterHydrogel-HLCs on acute liver failure in mice.

[0037] Figure 2 The results of observing the fluidity of the mixture of the dECM solution and the ODex solution, the dECM-ODex pregel, and the dECM-ODex hydrogel in Example 1 of the present invention, wherein A represents the mixture, B represents the dECM-ODex pregel, and C represents the dECM-ODex hydrogel;

[0038] Figure 3 This is a picture of the dECM-ODex hydrogel in Example 1 of the present invention;

[0039] Figure 4 FTIR spectra of Dex, ODex and dECM-ODex hydrogels in Example 1 of the present invention;

[0040] Figure 5 The rheological analysis results of dECM-ODex hydrogels with different n(NaIO4) / n(Dex) and different ODex concentrations in Example 2 of the present invention, A and B represent the storage modulus (G') and loss modulus (G") of the hydrogel during the frequency sweep rheological analysis, and C and D represent the average values ​​of G' and G" of the rheological analysis at 0.1 Hz and 1% strain;

[0041] Figure 6 The storage modulus (G') and loss modulus (G") of the Faster Softer hydrogel, the Slower Softer hydrogel and the Slower Stiffer hydrogel in Example 2 of the present invention;

[0042] Figure 7 The creep test results of Faster Softer hydrogel, Slower Softer hydrogel and SlowerStiffer hydrogel in Example 2 of the present invention, A represents the creep curve, B represents the creep response (% strain at t = 60s, n = 3);

[0043] Figure 8The compression test results of Faster Softer hydrogel, Slower Softer hydrogel, SlowerStiffer hydrogel and mouse liver in Example 3 of the present invention, A represents the initial elastic modulus, B represents the stress relaxation curve, and C represents the stress relaxation time scale τ 1 / 2 , D represents the stress relaxation percentage at 60 seconds;

[0044] Fig. 9 The SEM images of the Faster Softer hydrogel, the Slower Softer hydrogel and the SlowerStiffer hydrogel in Example 3 of the present invention;

[0045] Fig.10 The test results of the biocompatibility test of the dECM-ODex hydrogel in Example 4 of the present invention, A represents the live / dead staining result of hADSCs, and B represents the cell viability test result;

[0046] Fig.11 The results of the dECM-ODex hydrogel degradation test in Example 5 of the present invention, A represents the degradation in type II collagenase, and B represents the degradation in differentiation medium;

[0047] Fig.12 Compression test results of Faster Softer hydrogel, Slower Softer hydrogel and Slower Stiffer hydrogel after 23 days of cell differentiation in Example 6 of the present invention, A represents the initial elastic modulus, B represents the stress relaxation curve, and C represents the stress relaxation time scale τ 1 / 2 , D represents the stress relaxation percentage at 60 seconds;

[0048] Fig.13 The RT-qPCR analysis results of Faster Softer hydrogel, SlowerSofter hydrogel and Slower Stiffer hydrogel encapsulating cells after 23 days of differentiation in Example 6 of the present invention;

[0049] Fig.14 The immunofluorescence staining results (A, C) and quantitative analysis results (B, D) of Faster Softer hydrogel, SlowerSofter hydrogel and Slower Stiffer hydrogel encapsulating cells after 23 days of differentiation in Example 6 of the present invention are shown;

[0050] Fig.15 The results of the culture supernatant test of the differentiation culture for 23 days in Example 6 of the present invention;

[0051] Fig.16The results of the effects of Faster Softer-hADSC-CM, Slower Softer-hADSC-CM, and Slower Stiffer-hADSC-CM on HUVECs migration in Example 7 of the present invention, A represents the migration of HUVECs, B represents the quantitative analysis of 24h cell migration (n=3), and C represents the quantitative analysis of 72h cell migration (n=3), scale: 200μm;

[0052] Fig.17 The results of the effects of Faster Softer-HLC-CM, Slower Softer-HLC-CM, and SlowerStiffer-HLC-CM on HUVECs migration in Example 7 of the present invention, A represents the migration of HUVECs, and B represents the quantitative analysis of 24h and 72h cell migration (n=3), scale: 200μm;

[0053] Fig.18 The staining results (A) and quantitative analysis results (B) of Faster Softer-HLC-CM, Slower Softer-HLC-CM, and SlowerStiffer-HLC-CM promoting angiogenesis in Example 7 of the present invention, scale: 200 μm;

[0054] Fig.19 The results of the ROS scavenging ability of Faster Softer-HLC-CM, Slower Softer-HLC-CM, and SlowerStiffer-HLC-CM in Example 7 of the present invention, A represents the immunofluorescence imaging results, B represents the flow cytometry quantitative analysis results, scale: 100 μm;

[0055] Fig. 20 The anti-inflammatory effects of Faster Softer-HLC-CM, Slower Softer-HLC-CM, and SlowerStiffer-HLC-CM on Raw264.7 cells in Example 7 of the present invention, A represents the relative mRNA expression levels of pro-inflammatory marker genes (Il1b, Cox2, Tnf, and Nlrp3) in Raw264.7 cells after inflammatory stimulation (n=3); B represents the relative mRNA expression levels of anti-inflammatory marker genes Arg1 and Il10 in Raw264.7 cells after inflammatory stimulation (n=3); C represents the representative fluorescence images of CD86 (green) and CD206 (red) staining of Raw264.7 cells after inflammatory stimulation; Scale bar: 100 μm;

[0056] Fig.21The anti-inflammatory effects of Faster Softer-HLC-CM, Slower Softer-HLC-CM, and SlowerStiffer-HLC-CM on BMDMs in Example 7 of the present invention;

[0057] Fig. 22 The treatment effect of the mouse acute liver failure model in Example 8 of the present invention, A represents a schematic diagram of the in vivo treatment scheme for CCl4-induced ALF; B represents serum liver injury markers including transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and total bilirubin (TBiL1) analysis (n=3); C represents hematoxylin and eosin (H&E) staining images of liver sections, scale: 200μm; D represents quantitative analysis of necrotic areas (n=3); E represents immunofluorescence staining of hepatocyte marker human HNF4α in liver tissue of group V, scale: 50μm; F represents Tunel staining of liver sections; G represents quantitative analysis of Tunel-positive cells (red) (n=3), scale: 50μm; H represents Ki67 staining of liver sections; I represents corresponding Ki67-positive cells (red) quantitative analysis (n=3), scale: 50μm;

[0058] Fig.23 This is the liver morphology image in Example 8 of the present invention, scale: 0.5 cm;

[0059] Fig.24 The H&E staining results of the main organs (heart, spleen, lung, and kidney) of ALF mice in different treatment groups after transplantation in Example 8 of the present invention, scale: 200 μm;

[0060] Fig.25 The test results of the antioxidant and inflammatory levels of the liver of ALF model mice after different treatments in Example 8 of the present invention, A represents RT-qPCR analysis of the expression of liver function genes Cyp1a2, Cyp3a11, and Sox9 (n=3); B represents RT-qPCR detection of the expression of antioxidant genes Ho1, Gpx1, and Nqo1 in liver tissue (n=3); C represents RT-qPCR detection of the expression of inflammatory genes il-1b and Nlrp3 in liver tissue (n=3); D represents HO-1 immunofluorescence staining and quantitative analysis of liver sections (n=3), scale: 50μm; E represents TNF-α immunofluorescence staining and quantitative analysis of liver sections (n=3), scale: 50μm;

[0061] Fig.26 The results of CD31 immunofluorescence staining of liver sections in Example 8 of the present invention, scale: 50: μm. DETAILED DESCRIPTION

[0062] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0063] Example 1

[0064] 1. Preparation of decellularized matrix (dECM) pregel solution

[0065] Approximately 200 grams of pig liver obtained from the market was divided into small cubes of 1 cm × 1 cm × 1 cm to ensure that there was no connected tissue. The liver tissue was subjected to three cycles of freezing at -80°C and thawing at room temperature, and washed every 2 hours in ultrapure water. Subsequently, the liver tissue was immersed in an aqueous solution containing 1% Triton X-100, 0.1% ammonia solution, 1% penicillin-streptomycin and 10mM PMSF for 3 days, during which the solution was changed 3 times a day, and the sample was continuously stirred at 150 rpm for decellularization to obtain decellularized tissue. After this, the decellularized tissue was stirred and washed in ultrapure water containing 1% penicillin for three days, with fresh water changed three times a day, and then freeze-dried using a vacuum freeze dryer to obtain a dry decellularized matrix (dECM). The decellularized matrix (dECM) was further digested with pepsin (Sigma-Aldrich, USA), added to a 0.1 M hydrochloric acid aqueous solution at a weight ratio of dried dECM: pepsin (10:1), and stirred at room temperature for 48 h to prepare a dECM solution with a dECM concentration of 15 mg / mL.

[0066] 2. Synthesis of oxidized dextran (ODex)

[0067] First, dextran (Dex) was dissolved in deionized water to obtain a uniform 10% (w / v) Dex solution. Then, a 10% (w / v) sodium periodate (NaIO4) solution was gradually added to the dextran solution at a molar ratio of sodium periodate to dextran (n(NaIO4): n(Dex)) of 0.5:1. The reaction was carried out for 4 hours at 25°C in the dark. Subsequently, 4 mL of ethylene glycol was gradually added to stop the reaction. The resulting solution was then purified by dialysis against deionized water (MW = 3500) for at least 3 days to remove residual sodium periodate and other small molecules. Finally, ODex was obtained by freezing the purified solution and lyophilizing it using a vacuum freeze dryer (Scientz, China).

[0068] 3. Synthesis of decellularized matrix-oxidized dextran (dECM-ODex) hydrogel

[0069] The present invention utilizes the primary amine in the decellularized matrix to react with the aldehyde group of oxidized dextran to obtain the first layer network structure (pre-gel) of dECM-ODex hydrogel, and then polymerizes the collagen inside the decellularized matrix by reacting at 37°C to prepare a double-layer network dECM-ODex hydrogel. The schematic diagram of the preparation process of dECM-ODex hydrogel is shown in Figure 1 As shown in A.

[0070] Specifically, ODex was dissolved in DMEM basal medium to prepare an ODex solution. The pH of the 15 mg / mL dECM solution was adjusted to 7 and then immediately mixed with the ODex solution. The mixture was placed at room temperature for 30 minutes to form a preliminary hydrogel (pregel), and then incubated at 37°C for another 30 minutes to produce the final hydrogel (dECM-ODex hydrogel). The concentration of dECM in the resulting hydrogel was 10 mg / mL (1% w / v), and the final concentration of ODex was 0.25% w / v.

[0071] 3. Observation of hydrogel fluidity and structural integrity

[0072] The fluidity and structural integrity of the pregel and dECM-ODex hydrogel during the hydrogel preparation process were observed. The results showed that the pregel formed by the Schiff base reaction of ODex and dECM was weak, with high fluidity and weak strength, while the double-layer network dECM-ODex hydrogel prepared by further reaction (after incubation at 37°C for half an hour) had decreased fluidity and increased strength. The structural integrity of the dECM-ODex hydrogel was good.

[0073] The fluidity observation results of the mixture of dECM solution and ODex solution, dECM-ODex pregel and dECM-ODex hydrogel are shown in Figure 2 As shown in the figure, A represents the mixture, B represents the dECM-ODex pregel, and C represents the dECM-ODex hydrogel. Figure 3 shown.

[0074] 4. Spectrum test

[0075] The freeze-dried dECM-ODex hydrogel was crushed and mixed with KBr (Dex and ODex powders were directly mixed with KBr). Then, the test samples were prepared by the tablet method. Each sample was scanned 64 times using a Fourier transform infrared spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with a resolution range of 4000 cm -1 Up to 400cm -1 , and record the spectral data.

[0076] The FTIR spectra of Dex, ODex and dECM-ODex hydrogels are shown in Figure 4 As shown, the results showed that Dex was successfully oxidized to ODex, and a Schiff base reaction occurred between ODex and dECM, resulting in stable cross-linking within the hydrogel matrix.

[0077] Example 2

[0078] 1. Preparation of dECM-ODex hydrogels with different n(NaIO4):n(Dex) and different ODex concentrations

[0079] (1) Synthesis of oxidized dextran (ODex)

[0080] First, dextran (Dex) was dissolved in deionized water to obtain a uniform 10% (w / v) Dex solution. Then, 10% (w / v) sodium periodate (NaIO4) solution was gradually added to the dextran solution at different molar ratios of sodium periodate to dextran (n(NaIO4): n(Dex) was 0.5:1, 1.0:1 and 1.5:1, that is, 0.5, 1.0, 1.5). The reaction was carried out for 4 hours at 25°C in the dark. Subsequently, 4 mL of ethylene glycol was gradually added to stop the reaction. The resulting solution was then purified by dialysis against deionized water (MW = 3500) for at least 3 days to remove residual sodium periodate and other small molecules. Finally, ODex with different degrees of oxidation was obtained by freezing the purified solution and lyophilizing it using a vacuum freeze dryer (Scientz, China). The reaction of NaIO4 with Dex in different molar ratios (0.5:1, 1:1, 1.5:1) can produce ODex with different percentages of aldehyde groups, and ODex with different degrees of oxidation were synthesized.

[0081] (2) Synthesis of decellularized matrix-oxidized dextran (dECM-ODex) hydrogel

[0082] ODex with different molar ratios of sodium periodate to dextran were dissolved in DMEM basal culture medium to prepare ODex solutions of different concentrations with different degrees of oxidation. The pH of the 15 mg / mL dECM pregel solution prepared in Example 1 was adjusted to 7 and then immediately mixed with the ODex solution. The mixture was placed at room temperature for 30 minutes and then incubated at 37°C for another 30 minutes to obtain a dECM-ODex hydrogel. The dECM concentration in the resulting hydrogel was 10 mg / mL, and the final concentrations of ODex were 0.25% w / v, 0.5% w / v, and 1% w / v.

[0083] 2. Rheological analysis of hydrogel

[0084] Hydrogel discs with a thickness of 1mm-1.4mm were prepared and tested after swelling in DMEM medium for 24 hours. The hydrogel was placed between the parallel plates of the rheometer, and its mechanical behavior, including storage modulus (G') and loss modulus (G"), was recorded by frequency sweep measurement (0.1Hz-10Hz) at a constant strain (10%) at 25°C. Quantitative analysis of the G' and G" of the hydrogel can evaluate the elastic and viscous components of the hydrogel. The rheological analysis results of dECM-ODex hydrogels with different n(NaIO4) / n(Dex) and different ODex concentrations are shown in Figure 2. Figure 5 As shown, A and B represent the storage modulus (G') and loss modulus (G") of the hydrogel during the frequency sweep rheological analysis, and C and D represent the average values ​​of G' and G" of the rheological analysis at 0.1 Hz and 1% strain. The results showed that the storage modulus (G') and loss modulus (G") of dECM-ODex hydrogels with different n(NaIO4) / n(Dex) and different ODex concentrations were different, showing different viscoelastic properties.

[0085] Based on the evaluation of rheological data, three hydrogels with different mechanical properties were selected, namely FasterSofter hydrogel, SlowerSofter hydrogel and SlowerStiffer hydrogel. The final concentrations of n(NaIO4):n(Dex), ODex and dECM corresponding to FasterSofter hydrogel, SlowerSofter hydrogel and SlowerStiffer hydrogel are shown in Table 1 below.

[0086] Table 1

[0087]

[0088] The storage modulus (G') and loss modulus (G") of Faster Softer hydrogel, Slower Softer hydrogel and Slower Stiffer hydrogel are shown in Figure 2. Figure 6 The results show that the Faster Softer hydrogel has similar storage modulus to the Slower Softer hydrogel, the Slower Stiffer hydrogel has a significantly higher storage modulus than the two softer hydrogels, and the FasterSofter hydrogel has a significantly lower loss modulus than the latter two hydrogels with slower stress relaxation rates. The viscoelasticity and stiffness requirements of the three hydrogels meet the requirements and can be used to independently explore the effects of viscoelasticity and stiffness.

[0089] 3. Strain analysis of hydrogel

[0090] Hydrogel discs with a thickness of 1 mm to 1.4 mm were prepared and tested after swelling in DMEM medium for 24 hours. The hydrogel was placed between the parallel plates of the rheometer and a constant shear stress of 100 Pa was applied for creep testing, and the change of shear strain over time was recorded. The creep test results of Faster Softer hydrogel, Slower Softer hydrogel and Slower Stiffer hydrogel are shown in Figure 2. Figure 7 As shown, A represents the creep curve, and B represents the creep response (% strain at t=60s, n=3). The results show that the three hydrogels are viscoelastic structures.

[0091] Example 3

[0092] 1. Compression tests were performed on Faster Softer hydrogel, Slower Softer hydrogel, Slower Stiffer hydrogel and mouse liver.

[0093] dECM-ODex hydrogel discs with a thickness of 500 μm and a diameter of 6 mm were prepared and equilibrated in low-glucose DMEM medium for 24 h to promote hydrogel swelling. Compression tests were then performed using a Microforce Tester (EFL, China). In these tests, the hydrogels were compressed at a rate of 1 mm min -1 The hydrogels were subjected to compressive deformations ranging from 0% to 10% at a deformation rate of 10%. This 10% compressive strain was maintained for 300 seconds and the corresponding stress response was recorded as a function of time. To determine the initial elastic modulus, the stress-strain data were plotted over the compressive strain range with an initial slope between 5% and 10%. The experimental data were fitted to a linear model and the resulting slope represents the initial elastic modulus of the hydrogel. To quantify the stress relaxation behavior at a compressive strain of 10%, τ 1 / 2 (defined as the half-life when the stress drops to half of its initial value). After each test, the hydrogel samples were discarded. To measure the stiffness and stress relaxation of liver tissue, 500 μm thick mouse liver tissue sections were prepared. The same method as for the hydrogel samples was used to measure the stiffness and stress relaxation of 6 mm diameter discs from the liver sections.

[0094] The compression test results of Faster Softer hydrogel, Slower Softer hydrogel, Slower Stiffer hydrogel and mouse liver are shown in Figure 2. Figure 8 As shown, A represents the initial elastic modulus, B represents the stress relaxation curve, and C represents the stress relaxation time scale τ 1 / 2, D represents the percentage of stress relaxation at 60 seconds. The results showed that Slower Stiffer hydrogel, FasterSofter hydrogel and Slower Softer hydrogel had different stress relaxation rates and the same softer stiffness, and Slower Softer hydrogel and Slower Stiffer hydrogel had the same slower stress relaxation rate and different stiffness. And the stiffness of Faster Softer hydrogel and Slower Softer hydrogel were both around 2KPa, which is within the stiffness range of natural liver tissue. Although these dECM-ODex hydrogels have slower stress relaxation rates compared to mouse liver tissue, they provide a repeatable and adjustable biophysical and biochemical system.

[0095] 2. Scanning electron microscopy analysis was performed on Faster Softer hydrogel, Slower Softer hydrogel and Slower Stiffer hydrogel.

[0096] The Faster Softer hydrogel, Slower Softer hydrogel and Slower Stiffer hydrogel were quickly frozen with liquid nitrogen and then freeze-dried in a vacuum freeze dryer (Scientz, China). The microstructures of the three hydrogels were observed using a 5 kV scanning electron microscope (SEM) (Phenom, Thermo Fisher Scientific, USA).

[0097] SEM images of Faster Softer hydrogel, Slower Softer hydrogel and Slower Stiffer hydrogel are shown in Figure 2. Fig. 9 As shown, the results showed that the hydrogels exhibited a complex layered porous structure, while the harder hydrogels had tighter internal cross-linking and smaller pore sizes, while the two softer hydrogel groups had larger pores.

[0098] Example 4

[0099] The biocompatibility of dECM-ODex hydrogel was tested.

[0100] 1. Cell Encapsulation

[0101] First, ODex solution was prepared with serum-free medium containing 5% serum substitute. To prepare for cell encapsulation, human adipose-derived mesenchymal stem cells (hADSCs) were centrifuged and resuspended in ODex solution at a concentration of 1×10 6 Cells mL -1. Then, the hydrogel was prepared according to the method of Example 2, so as to achieve the encapsulation of cells in the hydrogel. The hydrogel-encapsulated cells were placed in a serum-free culture medium containing 5% serum substitute and 5% CO2, and cultured in an incubator at 37°C. The culture medium was replaced regularly every 2 days. The final concentration of dECM in the hydrogel corresponding to the hydrogel-encapsulated cells was 1% w / v, n(NaIO4):n(Dex) was 1.5:1, and the final concentrations of ODex were 0.25% w / v, 0.5% w / v and 1% w / v, respectively, and the corresponding encapsulated cells were recorded as 0.25%, 0.5% and 1%, respectively.

[0102] 2. Cell viability detection

[0103] The cytotoxicity of the hydrogel was determined by the CCK-8 method. After culturing the hydrogel-encapsulated cells in the above-mentioned serum-free medium for 48 hours, the medium was replaced with fresh medium containing 10% CCK8 (APExBIO, USA). Subsequently, incubation was carried out at 37°C in a 5% CO2 incubator under light-shielding conditions for 3 hours. 100 μL of the sample solution was transferred to a 96-well tissue culture plate for analysis. The absorbance at 450 nm was measured using a microplate reader. In addition, the viability of hADSCs encapsulated in dECM-ODex hydrogels was evaluated using a live / dead assay kit (Biyuntian, China), and the cells were observed using an inverted fluorescence microscope.

[0104] 3. Results

[0105] The biocompatibility test results of dECM-ODex hydrogel are as follows Fig.10 As shown, A represents the live / dead staining results of hADSCs, and B represents the cell viability test results. The live / dead staining results show that after 10 days of culture, most cells in the dECM-ODex hydrogels prepared with different ODex concentrations maintained good viability, with almost no dead cells. The cell viability test results showed that there was no obvious cytotoxicity after 48 hours of cell encapsulation in dECM-ODex hydrogels prepared with different ODex concentrations. This shows that dECM-ODex hydrogels have good biocompatibility.

[0106] Example 5

[0107] The degradability of dECM-ODex hydrogel was tested.

[0108] The dECM-ODex hydrogel was incubated with 500 μL 1× PBS (containing 20 μg / mL type II collagenase) at 37°C for 24 h, 48 h, and 72 h, and the degradation of the hydrogel was recorded by taking photos;

[0109] According to the encapsulation method of Example 4, human adipose-derived mesenchymal stem cells (hADSCs) were encapsulated using dECM-ODex hydrogels. The hydrogel-encapsulated cells were first incubated in low-glucose DMEM supplemented with 20 ng / mL epidermal growth factor (EGF) and 10 ng / mL basic fibroblast growth factor (bFGF) for two days; then differentiated for one week in low-glucose DMEM supplemented with 20 ng / mL hepatocyte growth factor (HGF), 10 ng / mL bFGF and 0.61 mg / mL nicotinamide, and then maintained for two weeks in low-glucose DMEM maturation medium supplemented with 20 ng / mL tumor inhibitor M (OSM), 1 mM dexamethasone and 1×ITS (ITS, i.e. insulin-transferrin-selenium), for a total of 23 days of differentiation, and the gel degradation was observed.

[0110] The results of dECM-ODex hydrogel degradation test are as follows Fig.11 As shown, A represents the degradation in type II collagenase, and B represents the degradation in differentiation medium. The results show that the dECM-ODex hydrogel can be degraded after incubation in type II collagenase for 24 hours, and is basically completely degraded after 72 hours; while the encapsulated hADSCs have basically no changes after 23 days of differentiation culture in differentiation medium, and are basically not degraded, indicating that the dECM-ODex of the present invention is biodegradable and can remain stable in the culture medium, and can be suitable for long-term cell culture.

[0111] Example 6

[0112] 1. Hepatic differentiation of human adipose-derived mesenchymal stem cells (hADSCs)

[0113] The hADSCs were encapsulated using the three hydrogels of Example 2: Faster Softer hydrogel, Slower Softer hydrogel and Slower Stiffer hydrogel. The encapsulation method was the same as that of Example 4.

[0114] The hADSCs encapsulated in the three hydrogels were induced to differentiate into the hepatic lineage using a cytokine cocktail method. Specifically, the hydrogel-encapsulated cells were first incubated in low-glucose DMEM supplemented with 20ng / mL epidermal growth factor (EGF) and 10ng / mL basic fibroblast growth factor (bFGF) for two days; then differentiated in low-glucose DMEM supplemented with 20ng / mL hepatocyte growth factor (HGF), 10ng / mL bFGF and 0.61mg / mL nicotinamide for one week, and then maintained in low-glucose DMEM maturation medium containing 20ng / mL tumor inhibitor M (OSM), 1mM dexamethasone and 1×ITS (ITS, insulin-transferrin-selenium) for two weeks, for a total of 23 days of differentiation.

[0115] 2. Compression test

[0116] The compression test was performed on the hydrogel after cell differentiation according to the compression test method of Example 3.

[0117] The compression test results of Faster Softer hydrogel, Slower Softer hydrogel and Slower Stiffer hydrogel after 23 days of cell differentiation are shown in Figure 2. Fig.12 As shown, A represents the initial elastic modulus, B represents the stress relaxation curve, and C represents the stress relaxation time scale τ 1 / 2 , D represents the percentage of stress relaxation at 60 seconds. The results show that the physical parameters of the three hydrogels with different mechanical properties remain stable and do not change during differentiation, which can be used to explore the effects of stress relaxation and stiffness on liver differentiation.

[0118] 3. RT-qPCR

[0119] RNA was extracted for qRT-PCR experiments. Total RNA was extracted from the hydrogels of encapsulated cells after differentiation using TRIzon reagent and reverse transcribed using HiFiScript cDNA synthesis kit according to the manufacturer's protocol. Real-time fluorescence quantitative PCR was performed using UltraSYBR mixture. All data were normalized to GAPDH values, and relative expression was calculated using the delta-delta Ct method.

[0120] The RT-qPCR analysis results of Faster Softer hydrogel, Slower Softer hydrogel and SlowerStiffer hydrogel encapsulating cells after 23 days of differentiation are shown in Fig.13 As shown, TCPs are stem cells for 2D liver differentiation on culture dishes. The results show that the expression of albumin (ALB), α-1-antitrypsin (A1AT), cytochrome P450 family 3 (CYP3A4) genes and T-box transcription factor 3 (TBX3) genes in hADSCs differentiated by Faster Softer hydrogels was significantly higher than that in cells in Slower Softer hydrogels and Slower Stiffer hydrogels. The 3D liver differentiation effect in the three groups of gels was better than conventional 2D differentiation.

[0121] 4. Immunofluorescence staining

[0122] After differentiation, the hydrogels encapsulating the cells were fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature (RT), washed twice with 1× PBS, and then blocked with 5% goat serum for 1 h. Afterwards, the cells were incubated with primary antibodies overnight at 4°C. The cells were then incubated with Alexa Fluor 488 goat anti-rabbit conjugated secondary antibodies (1:500 in 1× PBS / 0.3% Triton X-100) for 2 h at RT and washed twice with 1× PBS. After staining the nuclei with DAPI for 15 min at RT, the cells were imaged by inverted fluorescence microscopy.

[0123] Immunofluorescence staining results (A, C) and quantitative analysis results (B, D) of Faster Softer hydrogel, Slower Softer hydrogel and SlowerStiffer hydrogel encapsulating cells after 23 days of differentiation Fig.14 As shown, in the FasterSofter hydrogel 3D differentiated hADSCs, the fluorescence intensity of albumin (ALB) and ATP-binding cassette subfamily C member 2 (ABCC2) was the highest, indicating that these two proteins were highly expressed.

[0124] 5. Determination of culture supernatant

[0125] On the 23rd day of differentiation culture, albumin (ALB), urea (Urea) and hepatocyte growth factor (HGF) produced in the culture supernatant were measured using an albumin assay kit, a urea assay kit and an HGF ELISA kit, respectively.

[0126] The results of the culture supernatant test on day 23 of differentiation culture are as follows Fig.15 As shown, the results showed that the secretion of hepatocyte growth factor (HGF), albumin (ALB) and urea (Urea) in hepatocytes (HLCs) induced in Faster Softer hydrogel was significantly increased compared with that in Slower Softer hydrogel and Slower Stiffer hydrogel.

[0127] In summary, all three hydrogels can promote the hepatic differentiation of human adipose-derived mesenchymal stem cells (hADSCs), among which Faster Softer hydrogel has a stronger promoting effect.

[0128] Example 7

[0129] 1. Collection of Conditioned Medium

[0130] Human adipose-derived mesenchymal stem cells (hADSCs) were encapsulated in dECM-ODex hydrogels with different mechanical properties (Faster Softer hydrogel, Slower Softer hydrogel and Slower Stiffer hydrogel) and then cultured in stem cell serum-free medium for 3 days. The medium was collected, centrifuged, and the supernatant was sterile filtered to obtain conditioned medium 1 (CM-1). The conditioned medium corresponding to different hydrogels were respectively recorded as: Faster Softer-hADSC-CM, Slower Softer-hADSC-CM, Slower Stiffer-hADSC-CM, and stored at -20°C for future use. After hADSCs were encapsulated with hydrogels, they were differentiated and cultured for 23 days according to the differentiation method of Example 4. After hADSCs were differentiated into hepatocytes (HLCs) (differentiation conditions were the same as in Example 6), the culture medium was collected, centrifuged, and the supernatant was sterile filtered to obtain conditioned medium 2 (CM-2). The conditioned medium corresponding to different hydrogels were respectively recorded as: Faster Softer-HLC-CM, Slower Softer-HLC-CM, Slower Stiffer-HLC-CM, and stored at -20°C for future use.

[0131] 2. Effect of conditioned medium on migration of human umbilical vein endothelial cells (HUVECs)

[0132] HUVECs were cultured at 5 × 10 4 / well were seeded on a 24-well plate and cultured overnight. The cells were starved in serum-free medium for 24 hours, and then a central straight line was drawn at the bottom of multiple wells with a 1 mL sterile pipette tip. The cells were gently washed twice with 1×PBS to remove debris and cultured with a mixed medium containing 0.5% FBS and CM-1 or CM-2 in a 1:1 volume ratio. The remaining scratch area was imaged and measured by Image J software at 24 and 48 h. The characterization of cell migration was performed using the formula: Cell migration (%) = ((S0-St) / S0) × 100%, where S0 is the initial scratch area and St is the scratch area at 24 or 48 h.

[0133] The effects of Faster Softer-hADSC-CM, Slower Softer-hADSC-CM, and Slower Stiffer-hADSC-CM on HUVECs migration are shown in the following table. Fig.16As shown, A represents the migration of HUVECs, B represents the quantitative analysis of 24h cell migration (n=3), and C represents the quantitative analysis of 72h cell migration (n=3). Scale bar: 200μm. The results showed that the three hydrogel-encapsulated hADSCs conditioned medium 1 (CM-1) had a certain effect on promoting HUVECs migration, among which Faster Softer-hADSC-CM had a better effect on promoting migration.

[0134] The effects of Faster Softer-HLC-CM, Slower Softer-HLC-CM, and Slower Stiffer-HLC-CM on HUVECs migration are shown in the following table. Fig.17 As shown, A represents the migration of HUVECs, and B represents the quantitative analysis of cell migration at 24h and 72h (n=3), scale: 200μm. The results showed that the conditioned medium 2 (CM-2) after 3D liver differentiation of hADSCs encapsulated in three hydrogels had a certain effect on promoting the migration of HUVECs, among which Faster Softer-HLC-CM had a better effect on promoting migration.

[0135] 3. In vitro tube formation test

[0136] Add 100 μl of Matrigel to a 96-well plate and place in a 37°C incubator for 30 minutes to form a gel. Resuspend HUVECs in a 1:1 volume ratio of medium without FBS and CM-2 to form 3×10 cells per well. 4 The cells were seeded on the matrix gel of a 96-well plate at a density of 100 μg / well. The angiogenesis was observed under an inverted microscope every 3 hours. After confirming the formation of angiogenesis, the cells were stained with calcein.

[0137] The results of staining (A) and quantitative analysis (B) of Faster Softer-HLC-CM, Slower Softer-HLC-CM, and Slower Stiffer-HLC-CM promoting angiogenesis Fig.18 As shown, scale: 200μm. The results showed that the conditioned medium 2 (CM-2) after three hydrogels encapsulated hADSCs for 3D liver differentiation had a certain effect on promoting angiogenesis, among which Faster Softer-HLC-CM had a better effect on promoting angiogenesis. This shows that the stiffness of the hydrogel plays an important role in angiogenesis, while the viscoelasticity of the hydrogel has no significant difference.

[0138] 4. Antioxidant Effect

[0139] AML-12 cells were cultured at 5×10 4The cells were seeded at a density of 1:1 / well on a 24-well plate, cultured for 24 hours with a mixed medium containing complete medium and CM-2 at a volume ratio of 1:1, and then treated with H2O2 (500μM) to induce ROS production (i.e., positive control). After 12 hours, the medium containing H2O2 was discarded, and the cells were incubated with DCFH-DA (diluted with DMEM without fetal bovine serum) for 30 minutes. After rinsing with PBS three times, the intracellular ROS level was detected under a fluorescence microscope, and after trypsin digestion, it was quantitatively analyzed by flow cytometry.

[0140] The results of the ROS removal capabilities of Faster Softer-HLC-CM, Slower Softer-HLC-CM, and Slower Stiffer-HLC-CM are as follows Fig.19 As shown, A represents the results of immunofluorescence imaging, and B represents the results of flow cytometry quantitative analysis. Scale: 100 μm. The results showed that the fluorescence signals of the conditioned medium 2 (CM-2) corresponding to different hydrogels were reduced after treatment, indicating that they all have the ability to scavenge ROS. Faster Softer-HLC-CM has a stronger ability to scavenge ROS, and the intracellular ROS positive rate dropped from 89.15% to 28.23%. The ROS scavenging rates of Slower Softer-HLC-CM and Slower Stiffer-HLC-CM are similar, reaching 41.23% and 57.78% respectively.

[0141] 5. Anti-inflammatory ability

[0142] Use 100 ng / mL -1 Mouse mononuclear macrophage leukemia cells (Raw264.7 cells) and mouse primary macrophages (BMDMs) were treated with lipopolysaccharide (LPS, Sigma-Aldrich) for 24 h to stimulate M1 polarization. Subsequently, the medium containing LPS was replaced, and Raw264.7 cells and BMDMs were mixed with CM-2 (1:1) in fresh medium for 24 h. RAW264.7 cells and BMDMs were then collected and gene expression was analyzed by RT-qPCR. Immunofluorescence staining was used to detect the protein expression levels of M1 macrophage marker CD86 and M2 macrophage marker CD206 in RAW264.7 cells.

[0143] Anti-inflammatory effects of Faster Softer-HLC-CM, Slower Softer-HLC-CM, and Slower Stiffer-HLC-CM on Raw264.7 cells Fig. 20As shown, A represents the relative mRNA expression level of pro-inflammatory marker genes (Il1b, Cox2, Tnf and Nlrp3) in Raw264.7 cells after inflammatory stimulation (n=3); B represents the relative mRNA expression of anti-inflammatory marker genes Arg1 and Il10 in Raw264.7 cells after inflammatory stimulation (n=3); C represents the representative fluorescence image of CD86 (green) and CD206 (red) staining of Raw264.7 cells after inflammatory stimulation; scale: 100μm. The results showed that the conditioned medium 2 (CM-2) corresponding to different hydrogels may make mouse macrophages Raw264.7 susceptible to M2 polarization (anti-inflammatory phenotype), and FasterSofter-HLC-CM has a better effect.

[0144] Anti-inflammatory effects of Faster Softer-HLC-CM, Slower Softer-HLC-CM, and Slower Stiffer-HLC-CM on BMDMs Fig.21 As shown, the results showed that the expression of pro-inflammatory genes (Il1b, Tnf, Nos2) corresponding to Faster Softer-HLC-CM treatment was the lowest, and the expression of anti-inflammatory genes (Mrc1) was the highest. BMDM treated with Faster Softer-HLC-CM showed better M2 activation.

[0145] Example 8

[0146] Treatment of acute liver failure model in mice. All animal studies were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University (SYSU-IACUC-2024000738).

[0147] Six-week-old male C57BL / 6J mice were used for in vivo experiments. 5 μL / g (mouse body weight) of 30% flaxseed oil CCl4 was injected intraperitoneally into the mice to establish an acute liver failure (ALF) model. Seventy mice were randomly divided into 7 groups: (I) healthy control group (Healthy), (II) acute liver failure model group (ALF), (III) Faster Softer hydrogel group (Faster SofterHydrogel), (IV) Faster Softer hydrogel-hADSCs group (Faster Softer Hydrogel-hADSCs), i.e. Faster Softer hydrogel encapsulating hADSCs, (V) Faster Softer hydrogel-HLCs group (Faster SofterHydrogel-HLCs), i.e. Faster Softer hydrogel encapsulating hADSCs differentiated HLCs, (VI) Slower Softer hydrogel-HLCs group (Slower Softer Hydrogel-HLCs), i.e. Slower Softer hydrogel encapsulating hADSCs differentiated HLCs, (VII) Slower Stiffer Hydrogel-HLCs group (Slower Stiffer Hydrogel-HLCs), a Slow Stiffer hydrogel encapsulating hADSCs-differentiated HLCs.

[0148] Hydrogels containing or not containing cells were implanted in situ on the liver surface of ALF mice. The ALF group was treated with sham surgery. One day after transplantation, 0.6% sodium pentobarbital solution was injected intraperitoneally at a dose of 10 μL per gram of mouse body weight, and blood and organs were collected. The collected blood was allowed to stand at room temperature for 2 hours, centrifuged at 4°C for 30 minutes, and the supernatant was extracted to obtain serum. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total bilirubin (TBIL1) in serum were detected using AST detection kit, ALT detection kit, ALP detection kit, and TBIL1 detection kit, respectively, according to the manufacturer's protocol. The liver lobes were ground and RNA was extracted for RT-qPCR experiments. All tissues were preserved with 4% paraformaldehyde. Paraffin-embedded sections were subjected to hematoxylin and eosin (H&E) staining and immunofluorescence analysis.

[0149] Treatment effect of acute liver failure model in mice Fig. 22As shown, the results showed that Faster Softer Hydrogel-HLCs significantly alleviated the increase of ALT, AST and TBiL in CCl4-induced acute liver failure (Figure B). H&E staining showed that treatment with Faster Softer Hydrogel-HLCs could protect liver cells (Figure C) and significantly reduce the area of ​​injury from 60.98% to 19.95% (Figure D). This shows that Faster Softer Hydrogel-HLCs showed better liver protection compared with other treatment groups. Immunofluorescence staining of the hepatocyte marker human HNF4α in the liver tissue of group V confirmed that the hepatocyte-like cell HLCs encapsulated in the Faster Softer hydrogel were successfully implanted into the mouse liver (Figure E). Tunel staining results showed that the Tunel positive rate in the Faster Softer Hydrogel-HLCs treatment group was the largest compared with the modeling group, indicating that it effectively reduced hepatocyte apoptosis (Figures F, G). Ki67 staining results showed that all gel treatment groups had positive results, and the FasterSofter Hydrogel-HLCs treatment group had the highest positive rate, indicating that it has the best function in promoting hepatocyte proliferation (Figures H, I).

[0150] Liver morphology images Fig.23 As shown, scale bar: 0.5 cm, the results showed that the liver of the ALF group was edematous and pale. After treatment with hydrogels and hydrogels encapsulating different cells, the liver morphology was improved.

[0151] H&E staining results of major organs (heart, spleen, lung, and kidney) of ALF mice in different treatment groups after transplantation Fig.24 As shown, scale bar: 200 μm. The results showed that no obvious changes were observed in H&E staining of the mouse heart, spleen, lung and kidney after implantation of the encapsulated cell hydrogel, indicating that the hydrogel implant material of the present invention has no serious side effects and systemic toxicity.

[0152] Results of the detection of antioxidant and inflammatory levels in the liver of ALF model mice after different treatments Fig.25As shown, the results showed that the expression of liver function genes in mice implanted with Faster Softer Hydrogel-HLCs was significantly enhanced compared with other treatment groups, especially the Faster Softer Hydrogel-hADSCs group (Figure A), which was comparable to the healthy control group, indicating the effectiveness of Faster Softer Hydrogel-HLCs in promoting liver function recovery. Antioxidant gene expression was most significantly upregulated in ALF mice transplanted with Faster Softer Hydrogel-HLCs (Figure B). HO-1 immunofluorescence labeling showed that the intracellular expression level of Faster Softer Hydrogel-HLCs-treated mice was significantly higher than that of mice treated with other different hydrogel cells (Figure D), indicating that Faster Softer Hydrogel-HLCs can play an antioxidant role in mice with acute liver failure. Faster Softer Hydrogel-HLCs treatment resulted in a significant decrease in the levels of pro-inflammatory genes il-1b and Nlrp3 in the liver of ALF mice (Figure C). The level of inflammatory marker TNF-α in the Faster Softer Hydrogel-HLCs group was the lowest compared with the modeling group (Figure E), indicating that Faster Softer Hydrogel-HLCs treatment has the potential to alleviate the increase of proinflammatory cytokines and inhibit the inflammatory response.

[0153] The results of CD31 immunofluorescence staining of liver sections are as follows Fig.26 As shown, scale: 50:μm. The results showed that the incidence of IV microvessels was higher, which promoted the formation of new blood vessels in the body and further enhanced the therapeutic effect. This shows that Faster Softer hydrogel has a better effect.

[0154] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a hydrogel capable of regulating stem cell differentiation, characterized in that: The following steps are involved: S1. Mixing the dried decellularized matrix and protease and dissolving them in a hydrochloric acid solution to prepare a decellularized matrix solution; the mass ratio of the decellularized matrix to the protease is (5-20):1; S2, mixing sodium periodate and dextran and dissolving them in water, protecting from light for full reaction, and then adding ethylene glycol to terminate the reaction; dialysis purification to remove residual sodium periodate and other small molecules, and drying the purified solution to obtain oxidized dextran; the molar ratio of sodium periodate to dextran is (0.5-1.5):1; S3, dissolving oxidized dextran in a solvent to obtain an oxidized dextran solution, then mixing it with the decellularized matrix solution of step S1, obtaining a pregel through a Schiff base reaction, and incubating the pregel in an environment of 35 to 40° C. to obtain the hydrogel; the solvent includes water, PBS or cell culture medium; the mass ratio of the decellularized matrix to the oxidized dextran in the hydrogel is 1: (0.1 to 1).

2. The preparation method according to claim 1, characterized in that In step S1, the decellularized matrix includes a liver decellularized matrix, a heart decellularized matrix or a kidney decellularized matrix; And / or, the protease comprises pepsin, trypsin, papain or collagenase.

3. The preparation method according to claim 1, characterized in that: In step S2, the reaction temperature for the full reaction is 20°C to 30°C, and the reaction time is 3h to 5h.

4. The preparation method according to claim 1, characterized in that: In step S3, the reaction temperature of the Schiff base reaction is 20°C to 30°C, and the reaction time is 10min to 60min; And / or, the incubation time is 30 min to 60 min.

5. The hydrogel prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the hydrogel according to claim 5 in regulating stem cell differentiation.

7. Use of the hydrogel according to claim 5 in preparing a product for treating liver diseases.

8. Use of the hydrogel according to claim 5 in combination with stem cells in the preparation of a product for treating liver diseases.

9. The use according to claim 7 or 8, characterized in that The liver disease includes liver failure or liver fibrosis; And / or, the stem cells include mesenchymal stem cells, embryonic stem cells or human induced pluripotent stem cells.

10. A product for treating liver disease, characterized in that: Comprising the hydrogel according to claim 5.

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