A sodium alginate-based hydrogel and a method for preparing the same

Abstract

The application belongs to the technical field of hydrogel preparation, and particularly relates to a sodium alginate-based hydrogel and a preparation method thereof, which comprises raw materials, and the raw materials are composed of the following components in parts by weight: 20 mL of morpholine ethanesulfonic acid (MES) buffer, 0.06 g of N-hydroxysuccinimide (NHS), 0.2 g of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC), RGD polypeptide in a molar ratio of RGD:SA (50 mL, 2%) = 1:15 or 1:20, 175 muL of EDC / NHS activation solution, 1 g of sodium alginate (SA), 51 mL of phosphate buffer (PBS), 1.44 g of low-acylated gellan gum powder, 0.16 g of high-acylated gellan gum powder, 0.1 g of RGD-SA powder, 0.2 g of VN-SA powder, 0.1 g of QK-SA powder, VN polypeptide in a molar ratio of VN:SA = 1:15, and QK polypeptide in a molar ratio of QK:SA = 1:15. The application enhances the cell adhesion of the hydrogel, promotes the proliferation and differentiation of cells, has controllable mechanical strength and slow degradation characteristics, and is beneficial to long-term culture of cells and tissue repair.

Description

Technical Field

[0001] This invention relates to the field of hydrogel preparation technology, and in particular to a sodium alginate-based hydrogel and its preparation method. Background Technology

[0002] Sodium alginate (SA) is a natural polysaccharide with good biocompatibility, biodegradability, and mechanical properties, and is widely used in the biomedical field, such as tissue engineering, drug delivery, and cell culture. In recent years, with the development of tissue engineering and regenerative medicine, higher requirements have been placed on biomaterials used for cell culture and tissue repair.

[0003] In the prior art, traditional hydrogel materials lack cell adhesion sites on their surface, resulting in limited cell adhesion and proliferation capabilities in the hydrogel, making it difficult to meet the mechanical requirements of materials in tissue engineering, especially in long-term culture and complex physiological environments. To address this issue, we propose a sodium alginate-based hydrogel and its preparation method. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies, such as the lack of cell adhesion sites on the surface of traditional hydrogel materials, which limits the adhesion and proliferation capacity of cells in hydrogels and makes it difficult to meet the mechanical requirements of materials in tissue engineering, especially for applications in long-term culture and complex physiological environments. Therefore, this invention proposes a sodium alginate-based hydrogel and its preparation method.

[0005] This application provides a sodium alginate-based hydrogel and its preparation method, which adopts the following technical solution:

[0006] A sodium alginate-based hydrogel comprises raw materials, which are composed of the following components in parts by weight: 20 mL of morpholine ethanesulfonic acid (MES) buffer, 0.06 g of N-hydroxysuccinimide (NHS), 0.2 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), RGD peptide at a molar ratio of RGD:SA (50 mL, 2%) of 1:15 or 1:20, 175 μL of EDC / NHS activation solution, 1 g of sodium alginate (SA), 51 mL of phosphate buffer (PBS), and low-acylation gelling agent. 1.44g of gelatin powder, 0.16g of highly acylated gellan gum powder, 0.1g of RGD-SA powder, 0.2g of VN-SA powder, 0.1g of QK-SA powder, VN peptides in a VN:SA molar ratio of 1:15, QK peptides in a QK:SA molar ratio of 1:15, the specific sequence of RGD peptide is GGGRGDASSP-NH2, the specific sequence of VN peptide is KGGPQVTRGDVFTMP-NH2, and the specific sequence of QK peptide is KLTWQELYQLKYKGI-NH2.

[0007] This invention also proposes a method for preparing a sodium alginate-based hydrogel, wherein the sodium alginate-based hydrogel is the aforementioned sodium alginate-based hydrogel, comprising the following steps:

[0008] S1: N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to morpholine ethanesulfonic acid (MES) buffer to dissolve and prepare an activation solution, which was then filtered to remove bacteria.

[0009] S2: Dissolve RGD peptide, VN peptide and QK peptide in phosphate buffer to prepare peptide working solutions;

[0010] S3: Add sodium alginate to phosphate buffered saline (PBS) and stir to dissolve;

[0011] S4: Add the activation solution to the dissolved sodium alginate solution and stir. Then add the peptide working solution and stir to obtain peptide-modified functionalized sodium alginate RGD-SA, VN-SA and QK-SA solutions. After freeze-drying, obtain RGD-SA powder, VN-SA powder and QK-SA powder.

[0012] S5: Dissolve low-acylated gellan gum powder, high-acylated gellan gum powder, RGD-SA powder, VN-SA powder and QK-SA powder in deionized water in different proportions to obtain basic hydrogel uncrosslinked solution and organoid hydrogel uncrosslinked solution, and perform sterilization filtration in a clean bench.

[0013] S6: Mix the uncrosslinked solutions of the basic hydrogel and the organoid hydrogel with α-MEM culture medium in a certain proportion, and wait for the reaction to crosslink into gel to obtain the basic hydrogel and the organoid hydrogel.

[0014] Further, in step S1, a 0.1 mol / L morpholine ethanesulfonic acid (MES) buffer solution is prepared and the pH is adjusted to 6.0-7.5. 0.06 g of N-hydroxysuccinimide (NHS) and 0.2 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) are added to 20 mL of morpholine ethanesulfonic acid (MES) buffer solution and fully dissolved to prepare the activation solution. The solution is then filtered through a 0.22 μm filter in a clean bench and set aside for use.

[0015] Further, in step S2, the RGD peptide, the VN peptide, and the QK peptide are each dissolved in 1 mL of phosphate buffer at a molar ratio of RGD:SA (10 g) = 1:15, respectively, to prepare working peptide solutions.

[0016] Further, in step S3, 1g of sodium alginate (SA) is added to 50mL of phosphate buffer (PBS) and magnetically stirred (1000rpm, room temperature) for 4h to fully dissolve the sodium alginate (SA).

[0017] Further, in step S4, at room temperature, 175 μL of EDC / NHS activation solution is added to 50 mL of fully dissolved sodium alginate solution, magnetically stirred for 1 h, and then allowed to stand for 3 h to wait for sodium alginate activation. Then, 100 μL of the prepared peptide working solution is added to the activated sodium alginate solution, magnetically stirred for 1 h, to obtain peptide-modified functionalized sodium alginate RGD-SA, VN-SA and QK-SA solutions, which are transferred to a 4 °C refrigerator and allowed to stand for 20 h. After freeze-drying, RGD-SA powder, VN-SA powder and QK-SA powder are obtained.

[0018] Further, in step S4, the mixed solution is dialyzed for three days at 4°C using a dialysis bag (dialysis bag: molecular weight cutoff 3.5 kDa; dialysate: deionized water). The dialysate is changed every 6 hours on the first day, and every 12 hours on the second and third days. After dialysis, bacteria are removed by filtration, following the same procedure as above (only filtration through a 0.22 μm filter membrane is required). Then, RGD peptide-modified sodium alginate (RGD-SA) is prepared by aseptic freeze drying for later use. Under the same synthesis conditions, QK-modified sodium alginate (QK-SA) and VN-modified sodium alginate (VN-SA) are prepared.

[0019] Further, in step S5, 0.72g of low-acylated gellan gum powder, 0.08g of high-acylated gellan gum powder, 0.08g of RGD-SA powder, 0.10g of VN-SA powder and 0.02g of QK-SA powder are dissolved in 100mL of deionized water at 37°C to obtain a basic hydrogel uncrosslinked solution, which is then filtered and sterilized in a clean bench.

[0020] Further, in step S5, 0.72g of low-acylated gellan gum powder, 0.08g of high-acylated gellan gum powder, 0.02g of RGD-SA powder, 0.10g of VN-SA powder and 0.08g of QK-SA powder are dissolved in 100mL of deionized water at 37°C to obtain an organoid hydrogel uncrosslinked solution, which is then filtered and sterilized in a clean bench.

[0021] Further, in step S6, the uncrosslinked solution of the basic hydrogel is mixed with α-MEM culture medium at a ratio of 2:1, and the mixture is reacted for 5 minutes to allow crosslinking to form a gel, thereby obtaining the basic hydrogel; the uncrosslinked solution of the organoid hydrogel is mixed with α-MEM culture medium at a ratio of 2:1, and the mixture is reacted for 5 minutes to allow crosslinking to form a gel, thereby obtaining the organoid hydrogel.

[0022] In summary, this application includes at least one of the following beneficial technical effects:

[0023] This approach significantly enhances the cell adhesion of sodium alginate-based hydrogels by introducing peptides such as RGD, QK, and VN, promoting cell proliferation and differentiation, and significantly improving the proliferative capacity of osteoblasts, nerve cells, and angiogenic cells.

[0024] This invention enhances the cell adhesion of hydrogels, promotes cell proliferation and differentiation, and has controllable mechanical strength and slow degradation characteristics, which are beneficial for long-term cell culture and tissue repair. Attached Figure Description

[0025] Figure 1 This is a flowchart of a method for preparing sodium alginate-based hydrogel proposed in this invention;

[0026] Figure 2 (a, b, c, d, e) are schematic diagrams of the sodium alginate grafting polypeptide process in the preparation method of sodium alginate-based hydrogel proposed in this invention. Figure 2 a shows the reagents used in the process of grafting polypeptides (RGD, QK, VN) with sodium alginate. Figure 2 b is a schematic diagram of the process of activating sodium alginate solution using NHS / EDC activation solution. Figure 2 c and Figure 2Figure d shows the reaction conditions and time when the polypeptide solution is added to the activated sodium alginate (SA) solution at a molar ratio of 1:15. Figure 2 e is a schematic diagram of the dialysis process using a 3.5kDa dialysis bag at 4°C;

[0027] Figure 3 This is a fluorescence image obtained by fluorescence microscopy observation of a method for preparing sodium alginate-based hydrogel proposed in this invention, wherein ( Figure 3 a, b, and c) are fluorescence images of FITC-labeled peptide-grafted basic hydrogels. Figure 3 (d, e, f) are fluorescence images of FITC-labeled peptides grafted onto organoid hydrogels. Figure 3 (g, h, i) are fluorescence images of FITC-labeled, uncrosslinked sodium alginate solution RGD-sodium alginate (RGD-SA). Figure 3 j, k, l) are fluorescence images of the FITC-labeled, uncrosslinked sodium alginate solution QK-sodium alginate (QK-SA). Figure 3 m, n, o) are fluorescence images of uncrosslinked modified sodium alginate solution VN-sodium alginate (VN-SA) labeled with the fluorescent molecule FITC;

[0028] Figure 4 2% of the sodium alginate-based hydrogel preparation method proposed in this invention ( Figure 4 ac) and 3% ( Figure 4 df) Schematic diagram of the grafting rates of peptides (RGD, VN, and QK) in the uncrosslinked hydrogel solution;

[0029] Figure 5 This is a schematic diagram of the method for testing the relative viscosity of sodium alginate solution in the preparation method of sodium alginate-based hydrogel proposed in this invention. Figure 5 a) Schematic diagram of relative viscosity test results of sodium alginate solution at different time points (15 minutes, 5 days, 7 days). Figure 5 b);

[0030] Figure 6 This is a schematic diagram showing the pH values ​​of the basic hydrogel at different time points (15 minutes, 1 day, 3 days, and 5 days) of the preparation method of sodium alginate-based hydrogel proposed in this invention.

[0031] Figure 7 The basic hydrogel for the preparation method of sodium alginate-based hydrogel proposed in this invention is stored at room temperature. Figure 7 a) and 4℃ Figure 7 b) Save the permeability diagram one week later;

[0032] Figure 8The basic hydrogel for the preparation method of sodium alginate-based hydrogel proposed in this invention is stored at room temperature. Figure 8 a) and store at 4℃ ( Figure 8 b) Schematic diagram of reverse osmosis one week later;

[0033] Figure 9 The organoid hydrogel prepared by the sodium alginate-based hydrogel method proposed in this invention is stored at room temperature. Figure 9 a)) and store at 4℃ ( Figure 9 b) Permeability diagram for the following week;

[0034] Figure 10 The organoid hydrogel prepared by the sodium alginate-based hydrogel method proposed in this invention is stored at room temperature. Figure 10 a) and store at 4℃ ( Figure 10 b) Schematic diagram of reverse osmosis one week later;

[0035] Figure 11 The basic hydrogel for the preparation method of sodium alginate-based hydrogel proposed in this invention is stored at room temperature. Figure 11 a)) and store at 4℃ ( Figure 11 b) Schematic diagram of permeability and reverse osmosis two weeks later;

[0036] Figure 12 The organoid hydrogel prepared by the sodium alginate-based hydrogel method proposed in this invention is stored at room temperature. Figure 12 a) and store at 4℃ ( Figure 12 b) Schematic diagram of permeability and reverse osmosis two weeks later;

[0037] Figure 13 The basic hydrogel for the preparation method of sodium alginate-based hydrogel proposed in this invention is stored at room temperature. Figure 13 a)) and store at 4℃ ( Figure 13 b) Schematic diagram of permeability and reverse osmosis three weeks later;

[0038] Figure 14 The organoid hydrogel prepared by the sodium alginate-based hydrogel method proposed in this invention is stored at room temperature. Figure 14 a) and store at 4℃ ( Figure 14 b) Schematic diagram of permeability and reverse osmosis three weeks later;

[0039] Figure 15 The basic hydrogel for the preparation method of sodium alginate-based hydrogel proposed in this invention is stored at room temperature. Figure 15 a) and store at 4℃ ( Figure 15 b) Schematic diagram of permeability and reverse osmosis after four weeks;

[0040] Figure 16 The organoid hydrogel prepared by the sodium alginate-based hydrogel method proposed in this invention is stored at room temperature. Figure 16 a) and store at 4℃ ( Figure 16 b) Schematic diagram of permeability and reverse osmosis after four weeks;

[0041] Figure 17 (a, b, c) are schematic diagrams of the process of testing the rheological properties of sodium alginate-based hydrogels using a rotational rheometer (Thermo Hakke MARS3, USA) in the preparation method of sodium alginate-based hydrogels proposed in this invention.

[0042] Figure 18 The rheological property data of sodium alginate hydrogels for the preparation method of sodium alginate-based hydrogels proposed in this invention are shown in the figure. Figure 18 ae);

[0043] Figure 19 This is a graph showing the long-term stability data of a basic hydrogel dome stored at room temperature and 4°C for a method for preparing sodium alginate-based hydrogel proposed in this invention. Figure 19 ad);

[0044] Figure 20 This is a graph showing the long-term stability data of the basic hydrogel plate for the preparation method of sodium alginate-based hydrogel proposed in this invention, stored at room temperature and 4°C. Figure 20 ad);

[0045] Figure 21 (a, b, c, d) are rheological test data of the long-term stability of organoid hydrogel dome-type plates under storage conditions at room temperature and 4℃. Figure 22 Figure a shows the rheological properties of organoid hydrogel dome-type plates after storage at room temperature and 4°C for one week. Figure 22 Figure b shows the rheological properties of organoid hydrogel dome-type plates after storage at room temperature and 4°C for two weeks. Figure 22 c shows the rheological properties of organoid hydrogel dome-type plates stored at room temperature and 4°C for three weeks. Figure 22 d shows the rheological properties of organoid hydrogel dome-shaped plates after storage at room temperature and 4°C for four weeks.

[0046] Figure 22 (a, b, c, d) are schematic diagrams showing the long-term stability of rheological properties of organoid hydrogel plates prepared by the sodium alginate-based hydrogel preparation method proposed in this invention, stored at room temperature and 4°C. Figure 22 Figure a shows the rheological properties of organoid hydrogel plates after storage at room temperature and 4°C for one week. Figure 22 Figure b shows the rheological properties of organoid hydrogel plates after storage at room temperature and 4°C for two weeks. Figure 22c shows the rheological properties of the organoid hydrogel plate after storage at room temperature and 4°C for three weeks. Figure 22 d shows the rheological properties of organoid hydrogel plates after storage at room temperature and 4°C for four weeks.

[0047] Figure 23 This is a schematic diagram of the hydrogel filtration process in the preparation method of sodium alginate-based hydrogel proposed in this invention. Figure 23 a) Rheological results before hydrogel filtration ( Figure 23 b) and rheological data after hydrogel filtration ( Figure 23 c);

[0048] Figure 24 (ao) are digital photographs (ac, fh, km) and live / dead fluorescent staining images (d, e, i, j, n, o) of osteoblasts co-cultured with hydrogel for 1, 3, and 7 days according to the method for preparing sodium alginate-based hydrogel proposed in this invention, wherein the live / dead fluorescent dyes used are calcein-AM and propidium iodide.

[0049] Figure 25 (af) shows the 3D images of the hydrogel prepared by the sodium alginate-based hydrogel proposed in this invention and the live cell fluorescence staining on days 1, 3, and 7 of osteoblast culture, wherein the live cell fluorescence dye is calcein-AM;

[0050] Figure 26 The bar chart shows the cell proliferation results of osteoblasts co-cultured with the hydrogel on days 1, 3, and 7, according to the method for preparing sodium alginate-based hydrogel proposed in this invention.

[0051] Figure 27 (ai) are digital photos (a, b, e, f, i, j) and live / dead fluorescent staining images (c, d, g, h, k, l) of nerve cells co-cultured with hydrogel for 1, 3, and 7 days according to the method for preparing sodium alginate-based hydrogel proposed in this invention, wherein the live / dead fluorescent dyes used are calcein-AM and propidium iodide.

[0052] Figure 28 The bar chart shows the cell proliferation results of nerve cells co-cultured with the sodium alginate-based hydrogel on days 1, 3, and 7, according to the preparation method of sodium alginate-based hydrogel proposed in this invention.

[0053] Figure 29 (af) is a 3D image of the hydrogel prepared by the sodium alginate-based hydrogel proposed in this invention and the live cell fluorescence staining after 1, 3 and 7 days of nerve cell culture, wherein the live cell fluorescence dye is calcein-AM;

[0054] Figure 30(a, b, c) are rheological test results of nerve cells cultured in the dome-structured hydrogel of the sodium alginate-based hydrogel preparation method proposed in this invention on days 1, 3, and 7.

[0055] Figure 31 Digital photographs (a, b, e, f, ij) and live / dead fluorescent staining images (c, d, g, h, k, l) of angiogenic cells co-cultured with hydrogel for 1, 3, and 7 days, as proposed in this invention, are shown. The live / dead fluorescent dyes used are calcein-AM and propidium iodide.

[0056] Figure 32 The bar chart shows the cell proliferation results of angiogenic cells co-cultured with the sodium alginate-based hydrogel proposed in this invention on days 1, 3, and 7.

[0057] Figure 33 (af) shows the 3D images of the hydrogel and live cells cultured for 1, 3 and 7 days of angiogenic cells prepared by the method of sodium alginate-based hydrogel proposed in this invention, with the live cell fluorescent dye being calcein-AM.

[0058] Figure 34 Digital photographs and hydrogel rheological test results on day 1 after culturing the dome-structured hydrogel into vascular cells, as presented in this invention, according to the method for preparing sodium alginate-based hydrogels. Figure 34 a) Digital photograph and hydrogel rheological test results on day 3 after the dome-shaped hydrogel was cultured into vascular cells. Figure 34 b), Digital photograph of the dome-shaped hydrogel cultured into vascular cells on day 7 and hydrogel rheological test results ( Figure 34 c);

[0059] Figure 35 Digital photographs (a, b, e, f, i, j) and live / dead fluorescent staining images (c, d, g, h, k, l) of the hydrogel prepared by the sodium alginate-based hydrogel proposed in this invention after co-culturing with nerve cells and osteoblasts for 1, 3, and 7 days, respectively, are shown. The live / dead fluorescent dyes used are calcein-AM and propidium iodide.

[0060] Figure 36 The bar chart shows the cell proliferation results of the sodium alginate-based hydrogel prepared by the present invention on days 1, 3, and 7 of co-culturing with nerve cells and osteoblasts.

[0061] Figure 37Digital photographs (a, b, e, f, i, j) and live / dead fluorescent staining images (c, d, g, h, k, l) of the hydrogel prepared by the sodium alginate-based hydrogel proposed in this invention after co-culturing with nerve cells and angiogenic cells for 1, 3, and 7 days, respectively, are provided. The live / dead fluorescent dyes used are calcein-AM and propidium iodide.

[0062] Figure 38 The bar chart shows the cell proliferation results of the sodium alginate-based hydrogel prepared by the present invention on days 1, 3, and 7 of co-culturing with nerve cells and angiogenic cells.

[0063] Figure 39 Digital photographs (a, b, e, f, i, j) and live / dead fluorescent staining images (c, d, g, h, k, l) of the hydrogel prepared by the sodium alginate-based hydrogel method proposed in this invention after co-culturing osteoblasts and angiogenic cells for 1, 3, and 7 days, respectively, are shown. The live / dead fluorescent dyes used are calcein-AM and propidium iodide.

[0064] Figure 40 The bar chart shows the cell proliferation results of the sodium alginate-based hydrogel prepared by the present invention on days 1, 3, and 7 of co-culturing with osteoblasts and angiogenic cells. Detailed Implementation

[0065] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0066] Example

[0067] Reference Figure 1A sodium alginate-based hydrogel comprises raw materials, which are composed of the following components in parts by weight: 20 mL of morpholine ethanesulfonic acid (MES) buffer, 0.06 g of N-hydroxysuccinimide (NHS), 0.2 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), RGD peptides at a molar ratio of RGD:SA (50 mL, 2%) = 1:15 or 1:20, 175 μL of EDC / NHS activation solution, 1 g of sodium alginate (SA), 51 mL of phosphate buffer (PBS), and low-acylation gel. 1.44g of cold gel powder, 0.16g of highly acylated cold gel powder, 0.1g of RGD-SA powder, 0.2g of VN-SA powder, 0.1g of QK-SA powder, VN peptides in a VN:SA molar ratio of 1:15, QK peptides in a QK:SA molar ratio of 1:15, the specific sequence of RGD peptide is GGGRGDASSP-NH2, the specific sequence of VN peptide is KGGPQVTRGDVFTMP-NH2, and the specific sequence of QK peptide is KLTWQELYQLKYKGI-NH2.

[0068] This embodiment also proposes a method for preparing a sodium alginate-based hydrogel, wherein the sodium alginate-based hydrogel is the aforementioned sodium alginate-based hydrogel, and includes the following steps:

[0069] S1: N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to morpholine ethanesulfonic acid (MES) buffer to dissolve and prepare an activation solution, which was then filtered to remove bacteria.

[0070] S2: Dissolve RGD peptide, VN peptide and QK peptide in phosphate buffer to prepare peptide working solutions;

[0071] S3: Add sodium alginate to phosphate buffered saline (PBS) and stir to dissolve;

[0072] S4: Add the activation solution to the dissolved sodium alginate solution and stir. Then add the peptide working solution and stir to obtain peptide-modified functionalized sodium alginate RGD-SA, VN-SA and QK-SA solutions. After freeze-drying, obtain RGD-SA powder, VN-SA powder and QK-SA powder.

[0073] S5: Dissolve low-acylated gellan gum powder, high-acylated gellan gum powder, RGD-SA powder, VN-SA powder and QK-SA powder in deionized water in different proportions to obtain basic hydrogel uncrosslinked solution and organoid hydrogel uncrosslinked solution, and filter and sterilize them in a clean bench.

[0074] S6: Mix the uncrosslinked solutions of the basic hydrogel and the organoid hydrogel with α-MEM culture medium in a certain proportion, and wait for the reaction to crosslink into gel to obtain the basic hydrogel and the organoid hydrogel.

[0075] In this embodiment, in step S1, a 0.1 mol / L morpholine ethanesulfonic acid (MES) buffer solution was prepared and the pH was adjusted to 6.0-7.5. 0.06 g of N-hydroxysuccinimide (NHS) and 0.2 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were added to 20 mL of morpholine ethanesulfonic acid (MES) buffer solution and fully dissolved to prepare the activation solution. The solution was then filtered through a 0.22 μm filter in a clean bench and set aside for use.

[0076] In this embodiment, in S2, RGD peptide, VN peptide, and QK peptide are dissolved in 1 mL of phosphate buffer at a molar ratio of RGD:SA (10 g) = 1:15, respectively, to prepare working peptide solutions.

[0077] In this embodiment, in step S3, 1g of sodium alginate (SA) is added to 50mL of phosphate buffer (PBS) and magnetically stirred (1000rpm, room temperature) for 4h to fully dissolve the sodium alginate (SA).

[0078] In this embodiment, in step S4, at room temperature, 175 μL of EDC / NHS activation solution was added to 50 mL of fully dissolved sodium alginate solution. After magnetic stirring for 1 h, the solution was allowed to stand for 3 h to activate the sodium alginate. Then, 100 μL of the prepared peptide working solution was added to the activated sodium alginate solution, and the solution was magnetically stirred for 1 h to obtain peptide-modified functionalized sodium alginate solutions RGD-SA, VN-SA, and QK-SA. These solutions were then transferred to a 4°C refrigerator and allowed to stand for 20 h. After lyophilization, RGD-SA powder, VN-SA powder, and QK-SA powder were obtained. The mixed solution was dialyzed for three days at 4°C using a dialysis bag (dialysis bag: molecular weight cutoff 3.5 kDa; dialysate: deionized water). The dialysate was changed every 6 hours on the first day, and every 12 hours on the second and third days. After dialysis, bacteria were removed by filtration, following the procedure described above (only filtration through a 0.22 μm filter membrane was required). Then, RGD peptide-modified sodium alginate (RGD-SA) was prepared by aseptic freeze drying for later use. Under the same synthesis conditions, QK-modified sodium alginate (QK-SA) and VN-modified sodium alginate (VN-SA) were prepared.

[0079] In this embodiment, in step S5, 0.72g of low-acylated gellan gum powder, 0.08g of high-acylated gellan gum powder, 0.08g of RGD-SA powder, 0.10g of VN-SA powder and 0.02g of QK-SA powder are dissolved in 100mL of deionized water at 37°C to obtain a basic hydrogel uncrosslinked solution, which is then filtered and sterilized in a clean bench.

[0080] 0.72g of low-acylated gellan gum powder, 0.08g of high-acylated gellan gum powder, 0.02g of RGD-SA powder, 0.10g of VN-SA powder and 0.08g of QK-SA powder were dissolved in 100mL of deionized water at 37℃ to obtain an organoid hydrogel uncrosslinked solution, which was then filtered and sterilized in a clean bench.

[0081] In this embodiment, in step S6, the uncrosslinked solution of the basic hydrogel is mixed with α-MEM culture medium at a ratio of 2:1, and the mixture is reacted for 5 minutes to allow crosslinking to form a gel, thus obtaining the basic hydrogel. The uncrosslinked solution of the organoid hydrogel is mixed with α-MEM culture medium at a ratio of 2:1, and the mixture is reacted for 5 minutes to allow crosslinking to form a gel, thus obtaining the organoid hydrogel.

[0082] Experimental Example

[0083] I. Experimental Procedure

[0084] 1. Determination of sodium alginate-grafted peptides

[0085] To characterize the grafting performance of peptides in uncrosslinked hydrogel solutions and hydrogels, a certain amount of RGD-SA, QK-SA, VN-SA, basic hydrogels, and organoid hydrogels were first taken and FITC solution (0.5 mg / mL) was added. The mixture was stirred and reacted for 2 hours under light-protected conditions. After the reaction, the mixed solution was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and deionized water was used as the dialysate. Dialysis was performed at 4°C for 3 days to remove excess FITC. After dialysis, the fluorescence intensity of the samples was detected using a confocal microscope. The grafting effect of peptides was evaluated by the intensity of fluorescence.

[0086] The grafting rate of the peptides was quantitatively analyzed by nuclear magnetic resonance (NMR) (instrument model: Bruker Avance NEO600MHz, Germany). 10 mg of RGD-SA, QK-SA, and VN-SA were dissolved in deuterium water (D2O) and placed into NMR tubes. The NMR tubes were scanned using the rotor tube of the NMR spectrometer. Tetramethylsilane was used as the internal standard. 1 HNMR spectrum; 1In the HNMR spectrum, the peak at 3.7 ppm-4.1 ppm is the sodium alginate peak, while the peak at 2.88 ppm-2.89 ppm is the peptide peak. The peak areas of the two are calculated using formula (1), the molar ratio of sodium alginate to peptide is calculated using formula (2), and the peptide grafting rate is obtained using formula (3). The specific formulas are as follows:

[0087] Peak area = Peak height * Peak width (1)

[0088] Molar ratio = (peak area / molecular weight) ratio (2)

[0089] Grafting rate (%) = molar ratio of sodium alginate to polypeptide / 15 * 100% (3)

[0090] 2. Hydrogel permeability determination

[0091] Take a certain amount of uncrosslinked solution of either the basic hydrogel or the organoid hydrogel, and use phenol red-free α-MEM medium as a crosslinking agent. Mix them according to the gelation ratio, and then quickly add 300 μL of the mixture to a 24-well plate using a pipette. After gelation, cover the surface of the hydrogel with 300 μL of phenol red-containing α-MEM medium. At this point, the hydrogel is colorless, and the medium is pink. Store the 24-well plate in a 37°C constant temperature and humidity oven. Take photos at 0 h and 24 h to observe the penetration of small phenol red molecules from the upper medium into the lower SA hydrogel based on the color diffusion.

[0092] Take a certain amount of uncrosslinked solution of basic hydrogel or organoid hydrogel, use phenol red α-MEM medium as crosslinking agent, mix according to the gelation ratio, and quickly add 300 μL of the mixed solution to a 24-well plate using a pipette. After gelation, cover the surface of the hydrogel with 300 μL of phenol red-free α-MEM medium. At this time, the hydrogel is pink and the medium is colorless. Place the 24-well plate in a constant temperature and humidity oven at 37℃ and take pictures at 0h and 24h. Observe the penetration of phenol red small molecules from the lower SA hydrogel to the upper medium according to the color diffusion.

[0093] 3. Determination of hydrogel rheological properties

[0094] like Figure 17 As shown, rheological analysis of SA hydrogels was performed using a rotational rheometer (Thermo Hakke MARS3, USA). Different groups of hydrogels were prepared in 24-well plates. After full gelation, rheological tests were conducted at 37°C with a constant shear pressure of 1 Pa, a fixed plate distance of 1.0 mm, a scanning frequency of 0.1-10 Hz, and a shear rate of 0-100 rad / s. Angular frequency scanning was also performed to obtain rheological data. The specific volume ratios of hydrogels in the dome method and the flat plate method are as follows:

[0095]

[0096] 4. Three-dimensional cell culture

[0097] Cell suspensions were prepared using α-MEM medium (cell concentration 0.5-2 × 10⁶ cells / mL). 6 Cells / mL), the uncrosslinked solution of the basic or organoid hydrogel and the pipette tip were warmed at 37°C. The uncrosslinked hydrogel solution and cell suspension were added to a 5mL centrifuge tube at a 2:1 ratio and pipetted 5-10 times to mix thoroughly. Then, 25μL of the ungelled hydrogel mixture was quickly pipetted onto the center of a 96-well plate to form a dome structure. After waiting at room temperature for about 10 minutes to form a soft gel, the soft gel was covered with 100μL of culture medium to obtain a hydrogel containing cells (C-SA). The 96-well plate was placed in a cell culture incubator, and the medium was completely changed every 48 hours. 5. Cytotoxicity

[0098] After preparing C-SA according to step 3, the C-SA and cell suspension were cultured for 1 day, 3 days and 7 days respectively. The culture medium in the well plate was aspirated, and the cells were washed 2-3 times with PBS. Calcein-AM and propidium iodide (PI) working solution were added to each well (the volume was added according to the instructions). The cells were incubated at 37°C for 30 minutes. Live cells (yellow-green fluorescence) and dead cells (red fluorescence) were detected using a fluorescence inverted microscope.

[0099] After dissolving C-SA in 50 μL of deionized water in each well, add 50 μL of CCK-8 preparation solution (CCK-8: serum-free α-MEM medium = 1:5). Incubate in a cell culture incubator for 30 minutes, and then measure the absorbance (OD value) of each solution at 450 nm using a microplate reader. Perform CCK-8 assays at 1 day, 3 days and 7 days to calculate cell viability.

[0100] Microstructure observation of the dome: After C-SA was prepared according to step 3, the hydrogel was imaged on a confocal microscope.

[0101] II. Experimental Results

[0102] 1. Basic physicochemical characterization

[0103] Figure 2(ae) demonstrates the process of grafting polypeptides (RGD, QK, VN) with sodium alginate. First, an NHS / EDC activation solution was prepared and added to an SA solution. After stirring at room temperature for 1 hour, the solution was allowed to stand for 3 hours to complete the activation. Then, the polypeptide solution was added to the activated SA solution at a molar ratio of 1:15, and stirring was continued for 1 hour. The reaction was then carried out at 4°C for 20 hours. After the reaction was completed, the solution was dialyzed at 4°C for 3 days using a 3.5 kDa dialysis bag to remove unreacted small molecule impurities.

[0104] 1.1 Characterization of grafted peptides

[0105] like Figure 3 After 3 days of dialysis, the basic hydrogel ( Figure 3 ac), organoid hydrogels ( Figure 3 df) and uncrosslinked RGD-SA solution ( Figure 3 gi), QK-SA solution ( Figure 3 jl), VN-SA solution ( Figure 3 Both mo) exhibit a certain fluorescence intensity, indicating successful peptide grafting, but the grafting rate cannot be quantitatively detected; it needs to be calculated based on NMR results. Figure 4 The calculation results (ac) show that the grafting rates of 2% RGD-SA are 6.11%, 2% VN-SA are 6.26%, and 2% QK-SA are 5.46%. These grafting rates are all close to the theoretical value of 6.67%, indicating that a 2% sodium alginate solution concentration is a relatively ideal grafting concentration. Figure 4 (df) shows the grafting results of 3% sodium alginate peptides. Multiple impurity peaks appeared in the NMR spectrum, but based on the peak area ratio at the same position, the grafting rate of 3% RGD-SA was 6.44%, the grafting rate of 3% VN-SA was 6.5%, and the grafting rate of 3% QK-SA was 5.82%.

[0106] 1.2 Degradation performance of sodium alginate

[0107] Reference Figure 5 A significant negative correlation exists between the degradation of sodium alginate and its viscosity change. As sodium alginate degrades, the molecular chains break down, leading to a decrease in molecular weight and thus a reduction in the viscosity of the solution. This is confirmed by using an Ubbelohde viscometer. Figure 5 a) The viscosity of the hydrogel is tested to determine the degree of degradation of sodium alginate. Figure 5b) The relative viscosity was measured to be 68.64 after 15 minutes of preparation. After 5 days, the relative viscosity was measured to be 62.62, which was lower than the viscosity on the first day, indicating that sodium alginate had degraded. On the seventh day, the relative viscosity of the hydrogel was measured to be 60.69, which was lower than the relative viscosity on the fifth day, indicating that sodium alginate continued to degrade. However, the degradation behavior of sodium alginate was not obvious in the conventional cell culture process of about seven days and did not affect the cell culture process. 1.3 Hydrogel pH value

[0108] The pH value of hydrogels plays an important regulatory role in cell growth. An appropriate pH value is conducive to cell proliferation and differentiation, while excessively low or high pH values ​​may inhibit these processes or even damage cells. Figure 6 The data shows the pH value of the hydrogel at different time points after hydrogel formation. The pH value of the hydrogel was 7.2 immediately after gelation (15 minutes). The pH value of the hydrogel increased to 7.5 after 1 day of gelation. The data for 3 days and 5 days were 7.5, indicating that the pH value of the hydrogel was stable at 7.5, which is close to the pH value of 7.2-7.6 of the cell growth microenvironment. This shows that the pH value of the hydrogel is close to the pH value of the cell culture microenvironment, and the pH value is relatively stable over time, which is suitable for cell growth.

[0109] 1.4 Permeability of Hydrogels

[0110] The permeability of hydrogels is crucial in cell culture, directly affecting the transport of nutrients, oxygen, and metabolic waste, thereby influencing cell survival, proliferation, and function. The permeability and reverse osmosis of the basic and organoid hydrogels prepared in this protocol were investigated. The results demonstrated good permeability, and the permeability and reverse osmosis after storage at room temperature or 4°C for one month (four weeks) were verified.

[0111] The permeability test method for basic hydrogels and organoid hydrogels is as follows: Take out the colorless hydrogel and transfer it into a 24-well cell culture plate. Add an appropriate amount of pink culture medium containing phenol red to the surface of the hydrogel. The pink culture medium diffuses downward from the surface of the basic hydrogel. After 24 hours, observe whether the color is uniform throughout the well to determine the permeability.

[0112] The reverse osmosis test method for basic hydrogels and organoid hydrogels is as follows: Take out the pink hydrogel containing phenol red and transfer it into a 24-well cell culture plate. Add an appropriate amount of colorless culture medium without phenol red to the surface of the hydrogel. The pink culture medium diffuses upward from the bottom of the hydrogel in the well plate. After 24 hours, observe whether the color is uniform throughout the well to determine the reverse osmosis.

[0113] from Figure 7 It can be seen that at room temperature ( Figure 7 a) or 4℃ Figure 7b) After one week of storage under both storage conditions, the colorless basic hydrogel was removed and transferred to a 24-well cell culture plate. An appropriate amount of pink phenol red culture medium was added to the surface of the hydrogel. The pink culture medium diffused downwards from the surface of the basic hydrogel. After 24 hours, the color diffused evenly throughout the well, indicating that the basic hydrogel has good permeability after being stored at room temperature or 4°C for one week.

[0114] from Figure 8 It can be seen that at room temperature ( Figure 8 a) or 4℃ Figure 8 b) After being stored for one week under both storage conditions, the pink hydrogel containing phenol red was taken out and transferred to a 24-well cell culture plate. An appropriate amount of colorless culture medium without phenol red was added to the surface of the hydrogel. The pink culture medium diffused upward from the bottom of the hydrogel in the well plate, indicating that the basic hydrogel still has good reverse osmosis after being stored at room temperature or 4°C for one week.

[0115] from Figure 9 It can be seen that at room temperature ( Figure 9 a) or 4℃ Figure 9 b) After one week of storage under both storage conditions, the colorless organoid hydrogel was removed and transferred to a 24-well cell culture plate. An appropriate amount of pink phenol red culture medium was added to the surface of the hydrogel. The pink culture medium diffused downward from the surface of the basic hydrogel. After 24 hours, the color diffused evenly throughout the well, indicating that the organoid hydrogel still has good permeability after one week of storage at room temperature or 4°C.

[0116] from Figure 10 It can be seen that at room temperature ( Figure 10 a) or 4℃ Figure 10 b) After one week of storage under both storage conditions, the pink hydrogel containing phenol red was taken out and transferred to a 24-well cell culture plate. An appropriate amount of colorless culture medium without phenol red was added to the surface of the hydrogel. The pink culture medium diffused upward from the hydrogel at the bottom of the well plate, indicating that the organoid hydrogel still has good reverse osmosis after one week of storage at room temperature or 4°C.

[0117] from Figure 11 It can be seen that at room temperature ( Figure 11 a) or 4℃ Figure 11 b) After two weeks of storage under both storage conditions, the culture medium and the basic hydrogel in the well plate diffused evenly after 24 hours, indicating that the basic hydrogel still has good permeability and reverse osmosis after being stored at room temperature or 4°C for two weeks.

[0118] from Figure 12 It can be seen that at room temperature ( Figure 12 a) or 4℃ Figure 12b) After two weeks of storage under both storage conditions, the color of the culture medium and organoid hydrogel in the well plate was uniformly diffused after 24 hours, indicating that the organoid hydrogel still has good permeability and reverse osmosis after two weeks of storage at room temperature or 4°C.

[0119] from Figure 13 It can be seen that at room temperature ( Figure 13 a) or 4℃ Figure 13 b) After three weeks of storage under both storage conditions, the culture medium and the basic hydrogel in the well plate diffused evenly after 24 hours, indicating that the basic hydrogel still has good permeability and reverse osmosis after being stored at room temperature or 4°C for three weeks.

[0120] from Figure 14 It can be seen that at room temperature ( Figure 14 a) or 4℃ Figure 14 b) After three weeks of storage under both storage conditions, the color of the culture medium and organoid hydrogel in the well plate was uniformly diffused after 24 hours, indicating that the organoid hydrogel still has good permeability and reverse osmosis after three weeks of storage at room temperature or 4°C.

[0121] from Figure 15 It can be seen that at room temperature ( Figure 15 a) or 4℃ Figure 15 b) After four weeks of storage under both storage conditions, the culture medium and hydrogel color diffused evenly in the well plate after 24 hours, indicating that the basic hydrogel still has good permeability and reverse osmosis after four weeks of storage at room temperature and 4℃.

[0122] from Figure 16 It can be seen that at room temperature ( Figure 16 a) or 4℃ Figure 16 (b) After four weeks of storage under both conditions, the culture medium and hydrogel showed uniform color diffusion after 24 hours, indicating that the organoid hydrogel still exhibited good permeability and reverse osmosis after four weeks of storage at room temperature and 4°C. This demonstrates that both hydrogels possess good permeability and reverse osmosis under both storage conditions, and these properties do not change with prolonged storage time.

[0123] 2. Rheological properties

[0124] 2.1 Sodium alginate hydrogel

[0125] Figure 17 This describes the testing process for the rheological properties of hydrogels. The instrument used is a Thermo Hakke MARS3 rotational rheometer, and the rotor used is a P20TiL. Figure 17As shown in (ac), the hydrogel was placed at the center of the lower rotor, the height of the upper rotor was adjusted so that the distance between the upper rotor and the lower rotor was 1 mm, and then the rheological behavior of the hydrogel was tested by computer program control.

[0126] Figure 18 Sodium alginate hydrogel in the dome and paving ( Figure 18 a) Rheological test data at 15 minutes of gelation under both molding methods. The results show that the modulus of the dome and the pavement hydrogel is relatively stable in the 0-10Hz frequency range, but the modulus data becomes chaotic when the frequency exceeds 10Hz. Given that frequencies exceeding 10Hz are extremely rare in actual operation, data with frequencies greater than 10Hz will be ignored in subsequent analysis. The two sets of rheological test results for the dome hydrogel show that its modulus is between 20-30 Pa. Figure 18 bc), while the two sets of test data for the plated hydrogel indicate that its modulus is approximately 100 Pa ( Figure 18 (de) It can be seen that the strength of the planar hydrogel is significantly higher than that of the dome hydrogel. However, the modulus of both hydrogels is in the range of 10-200 Pa, which is considered not to have an adverse effect on cell growth.

[0127] 2.2 Long-term stability of sodium alginate hydrogel

[0128] Figure 19 The data are rheological test results regarding the long-term stability of the basic hydrogel dome under storage conditions at room temperature and 4°C. At room temperature, the basic hydrogel dome showed stability from week one to week four. Figure 19 The storage modulus (G') of the basic hydrogel dome remained within the range of 20-50 Pa and was relatively stable with frequency changes. When stored at 4℃, the basic hydrogel dome maintained its storage modulus from the first to the fourth week. Figure 19 The storage modulus (G') of the ad) remained at 10-20 Pa and was relatively stable at frequencies of 0.1-10 Hz. Comparing the storage modulus (G') of the hydrogels stored at room temperature and 4℃, it was found that the storage modulus (G') of the basic hydrogel dome stored at 4℃ was slightly lower than that stored at room temperature, but the hydrogels stored at the same temperature had better stability.

[0129] Figure 20 These are rheological test data regarding the long-term stability of the basic hydrogel plank under storage conditions at room temperature and 4°C. At room temperature, the basic hydrogel plank showed stability from week one to week four. Figure 20 The storage modulus (G') of the basic hydrogel plates remained within the range of 50-200 Pa, and G' was relatively stable with frequency during this process; when stored at 4℃, the storage modulus of the basic hydrogel plates was stable from the first to the fourth week. Figure 20The storage modulus (G') of the ad) remained at 10-100 Pa and was relatively stable at frequencies of 0.1-10 Hz. Comparing the storage modulus (G') of the hydrogel stored at room temperature and 4℃, it was found that the storage modulus (G') of the basic hydrogel plate stored at 4℃ was slightly lower than that stored at room temperature, but the basic hydrogel plate stored at both temperatures had good stability.

[0130] Figure 21 (a, b, c, d) show the rheological test data of the long-term stability of organoid hydrogel dome-shaped plates under storage conditions at room temperature and 4°C. Under storage conditions at room temperature, the rheological properties of the organoid hydrogel dome are relatively stable with frequency variation. Figure 21 In the first week, its storage modulus (G') was approximately 100 Pa, but by the fourth week ( Figure 21 At time d), the storage modulus (G') decreased to about 20 Pa. Although the strength was reduced, it was still within the strength range suitable for cell culture (10-200 Pa). Under the storage condition of 4℃, the storage modulus (G') of the organoid hydrogel dome remained at 10-20 Pa from the first to the fourth week, and showed good stability in the frequency range of 0.1-10 Hz. Comparing the rheological data under the two storage conditions, it can be found that the storage modulus (G') of the hydrogel stored at 4℃ is lower than that of the hydrogel stored at room temperature, but the hydrogel stored under the same temperature conditions has better stability.

[0131] Figure 22 (a, b, c, d) show the rheological test data of the long-term stability of organoid hydrogel planar plates under storage conditions at room temperature and 4℃. Under room temperature storage, the storage modulus (G') of the organoid hydrogel planar plates remained in the range of 50-200 Pa from the first to the fourth week, with little change. During this process, the storage modulus (G') changed relatively stably with frequency. Under 4℃ storage, the storage modulus (G') of the organoid hydrogel planar plates remained in the range of 10-100 Pa from the first to the fourth week. Similarly, like the organoid hydrogel planar plates stored at room temperature, it was relatively stable within four weeks and also relatively stable at frequencies of 0.1-10 Hz. Comparing the storage modulus (G') of the hydrogel under room temperature and 4℃ storage conditions, it was found that the storage modulus (G') of the organoid hydrogel planar plates stored at 4℃ was slightly lower than that stored at room temperature. However, the organoid hydrogel planar plates stored at both temperatures had good stability.

[0132] 2.3 Rheological properties before and after hydrogel filtration

[0133] Figure 23 b represents the rheological data before hydrogel filtration. Figure 23c represents the rheological data after hydrogel filtration. The amount of hydrogel filtered in one pass was 600-700 μL. The rheological data shows that the storage modulus (G') of the hydrogel dome before and after filtration is similar and can still be maintained at around 20 Pa. This indicates that filtration does not significantly change the gelation properties of the hydrogel.

[0134] 3. Cell experiments

[0135] 3.1 BMSCs

[0136] Figure 24 (ae, fI, ko) represent digital photographs and live / dead staining images of osteoblasts co-cultured in hydrogel for 1, 3, and 7 days, respectively. The green fluorescent portion represents calcein-AM entering the cells and being hydrolyzed by endogenous esterases in living cells, indicating live cells. The red fluorescent portion represents propidium iodide binding to the cell nucleus, indicating dead cells. Figures (24b, g, if) and (24c, h, m) show that after 1, 3, and 7 days of co-culture, the intensity of green fluorescence within the hydrogel increases with increasing culture time, indicating that the number of live cells is significantly greater than that of dead cells. The gradual increase in green fluorescence intensity within the hydrogel with prolonged culture time demonstrates its effective promotion of osteoblast proliferation. Figure 24 As can be seen from the digital photos in (a, f, k), co-culturing with osteoblasts does not destroy the shape of the hydrogel dome;

[0137] Figure 25 (b, d, f) are 3D images of live cell staining after co-culturing osteoblasts with hydrogel for different days. It can be seen that the intensity of green fluorescence increases significantly with the extension of culture time, indicating that it effectively promotes osteoblast proliferation.

[0138] Figure 26 To assess the cell proliferation of osteoblasts co-cultured with hydrogel for 1, 3, and 7 days, the CCK-8 assay was used to quantitatively detect cell proliferation in the hydrogel group and the two-dimensional cell group at days 1, 3, and 7, with the two-dimensional cell group as a control. It can be seen that the cell proliferation in the hydrogel group was higher than that in the two-dimensional cell group at all three time points. As the culture time increased, the ability of hydrogel to promote osteoblast proliferation first weakened and then strengthened. When co-cultured for 7 days, the ability of hydrogel to promote osteoblast proliferation was significantly enhanced compared with the two-dimensional cell group.

[0139] 3.2 Nerve Cells (RSC96)

[0140] Figure 27 (al) shows the live and dead staining images of hydrogel and nerve cells co-cultured for different days. At 1, 3 and 7 days of co-culture, the green fluorescence intensity of the hydrogel group was higher than that of the red fluorescence, indicating that the number of live cells was much greater than that of dead cells. As the culture time was extended, the green fluorescence intensity of the hydrogel group gradually increased, indicating that it effectively promoted the proliferation of nerve cells.

[0141] Figure 28 The study demonstrated cell proliferation after co-culturing hydrogel with nerve cells for 1, 3, and 7 days. The results showed that, compared with the two-dimensional culture surface, the proliferation rate of the hydrogel group was greater than that of the two-dimensional culture surface. As the culture time increased, the promoting effect of hydrogel on nerve cell proliferation gradually increased. On the 7th day of co-culture, the hydrogel group significantly enhanced the ability to promote nerve cell proliferation compared with the two-dimensional cell group.

[0142] Figure 29 (b, d, f) show 3D images of live-cell staining after different days of co-culturing with hydrogel and nerve cells. The results show that the green fluorescence intensity significantly increased from day 1 to day 3, indicating that the hydrogel effectively promoted nerve cell proliferation during this stage. However, the change in green fluorescence intensity was not significant from day 3 to day 7. This may be because nerve cells migrated during the culture process, leading to changes in cell distribution, which masked the fluorescence intensity differences caused by proliferation. Therefore, although the hydrogel significantly promoted nerve cell proliferation in the early stage, the fluorescence intensity difference between day 3 and day 7 was not significant due to the influence of cell migration.

[0143] Figure 30 Rheological data for nerve cells cultured on hydrogels on days 1, 3, and 7. Figure 30 a represents the storage modulus (G') of the hydrogel after culturing nerve cells in hydrogel on day 1, measured using a rotational rheometer. The storage modulus of the hydrogel was approximately 15 Pa. Figure 30 b represents the storage modulus (G') of the hydrogel after culturing nerve cells in hydrogel on day 3, measured by rotational rheometer, which is around 40-50 Pa. Figure 30 c represents the storage modulus (G') of the hydrogel after 7 days of culturing nerve cells in the hydrogel, which is close to 100 Pa using a rotational rheometer. The results show that the storage modulus of the hydrogel containing nerve cells gradually increases with the increase of cell culture time. The storage modulus is used to describe the strength of the hydrogel. That is, the strength of the hydrogel containing cells gradually increases with the increase of cell culture time. This may be because nerve cells in the hydrogel will proliferate and migrate over time, slightly increasing the strength of the hydrogel, but the increasing trend is not obvious.

[0144] 3.3 Angiogenic cells (HUVECs)

[0145] Figure 31(al) shows live and dead cell staining images of hydrogel and angiogenic cells co-cultured for different days. At days 1, 3, and 7, the green fluorescence intensity in the hydrogel group was higher than the red fluorescence, indicating that the number of live cells was significantly greater than that of dead cells. As the culture time increased, the green fluorescence intensity in the hydrogel group gradually increased, indicating that it effectively promoted angiogenic cell proliferation. However, at day 7, the green fluorescence intensity was relatively weaker than on day 3, and damage appeared at the edge of the dome. This indicates that at longer culture times (greater than 3 days), angiogenic cells have more synapses, and during proliferation, they disrupt the hydrogel dome structure, causing some cells to migrate out of the hydrogel, thus resulting in less significant cell proliferation.

[0146] Figure 32 To assess cell proliferation during co-culturing of hydrogel and angiogenic cells on days 1, 3, and 7, the CCK-8 assay was used to quantitatively detect cell proliferation in both the hydrogel and two-dimensional cell groups, with the two-dimensional cell group serving as a control. The results showed that the hydrogel's ability to promote angiogenic cell proliferation gradually increased with prolonged culture time, although the proliferation rate slowed slightly from days 1 to 3. This may be because after 3 days of culture, some angiogenic cells began to migrate from the hydrogel surface and escape from the hydrogel dome region, potentially leading to cell loss during subsequent medium changes and thus a decrease in cell proliferation rate. When the co-culture time reached 3 days, the hydrogel's ability to promote angiogenic cell proliferation was significantly enhanced compared to the two-dimensional cell group.

[0147] Figure 33 (af) shows 3D images of live cell staining after different culture days of hydrogel and angiogenic cells. It can be seen that the green fluorescence intensity significantly increases with prolonged culture time, indicating that it effectively promotes angiogenic cell proliferation. When the culture time exceeds 3 days, the green fluorescence intensity of the hydrogel decreases, indicating that the hydrogel cannot promote angiogenic cell proliferation. This result is consistent with... Figure 34 The analysis results are consistent;

[0148] Figure 34 These are rheological test data on days 1, 3, and 7 after culturing vascular cells in hydrogels. Figure 34 a represents day 1, and the storage modulus (G') of the hydrogel is around 15 Pa. Figure 34 When b is the third day, the storage modulus G' of the hydrogel is about 30 Pa. Figure 34 When c is day 7, the storage modulus G' of the hydrogel is close to 40 Pa. Therefore, the strength (storage modulus) of the hydrogel increases slightly with time. This is because angiogenic cells in the hydrogel will proliferate and migrate with the extension of culture time, but the increase in the strength of the hydrogel is not significant.

[0149] 3.4 Nerve cells (RSC96) and osteoblasts (BMSCs)

[0150] Figure 35 (al) shows the live and dead staining images of hydrogel co-cultured with nerve cells and osteoblasts for 1, 3, and 7 days. At 1, 3, and 7 days of co-culture, the green fluorescence intensity of the hydrogel group was higher than that of the red fluorescence, indicating that the number of live cells was much greater than that of dead cells. As the culture time was extended, the green fluorescence intensity of the hydrogel group gradually increased, indicating that it effectively promoted the proliferation of nerve cells and osteoblasts.

[0151] Figure 36 The cell proliferation of nerve cells and osteoblasts co-cultured with hydrogel for 1, 3, and 7 days was compared with that of two-dimensional culture surface as a control. It can be seen that the proliferation rate of the hydrogel group is greater than that of the two-dimensional culture surface. As the culture time is extended, the ability of hydrogel to promote cell proliferation gradually increases. When the co-culture time reaches 7 days, the ability of hydrogel to promote cell proliferation is significantly enhanced compared with the two-dimensional cell group.

[0152] 3.5 Nerve cells (RSC96) and angiogenic cells (HUVEC)

[0153] Figure 37 Images showing the liveness and deadness staining of hydrogels co-cultured with nerve cells and angiogenic cells for 1, 3, and 7 days. The green fluorescence intensity of the hydrogel group at days 1, 3, and 7 is also shown. Figure 37 a, c, e, g, i, k) are higher than red fluorescence ( Figure 37 b, d, f, h, j, l) indicate that the number of live cells is much greater than that of dead cells. As the culture time increases, the intensity of green fluorescence in the hydrogel group gradually decreases, indicating that when the culture time is long (greater than 3 days), there are more synapses in the angiogenic cells. During proliferation, the hydrogel dome structure is destroyed, causing some cells to crawl out of the hydrogel. The hydrogel structure is destroyed, so the cell proliferation effect is not obvious.

[0154] Figure 38 The cell proliferation of neuronal and angiogenic cells co-cultured with hydrogel for 1, 3, and 7 days was compared with the two-dimensional cell group as a control. It can be seen that the proliferation rate of the hydrogel group was lower than that of the two-dimensional cell group. This may be due to factors such as… Figure 37 As shown in a and 37i, hydrogel breakage occurred, and this increased with prolonged culture time. While hydrogels can promote cell proliferation, the proliferation capacity is significantly lower than in the two-dimensional cell group due to hydrogel rupture and cell migration.

[0155] 3.6 Bone stromal cells (BMSCs) and angiogenic cells (HUVECs)

[0156] Figure 39 Images showing the liveness and deadness staining of hydrogels co-cultured with osteoblasts and angiogenic cells for 1, 3, and 7 days. The green fluorescence intensity of the hydrogel group at days 1, 3, and 7 is also shown. Figure 39 a, c, e, g, i, k) are higher than red fluorescence ( Figure 39 (b, d, f, h, j, l) indicates that the number of live cells is far greater than that of dead cells. As the culture time increases, the intensity of the green fluorescence in the hydrogel group gradually decreases, indicating that it inhibits cell proliferation. This is because angiogenic cells have more synapses, and their proliferation disrupts the hydrogel dome structure, resulting in hydrogel structural damage.

[0157] Figure 40 This study compared cell proliferation in hydrogel-co-cultured osteoblasts and angiogenic cells for 1, 3, and 7 days, with the control group being a two-dimensional culture surface. The results showed that on day 1 of co-culture, the proliferation rate in the hydrogel group was significantly higher than that in the two-dimensional culture surface. However, as the culture time increased, the promoting effect of the hydrogel on cell proliferation significantly weakened. This phenomenon may be because the hydrogel begins to break down after one day of culture, allowing cells to emerge from the hydrogel dome structure.

[0158] 4. Conclusion

[0159] This invention successfully developed polypeptide-modified composite hydrogels (RGD-SA, QK-SA, VN-SA) based on sodium alginate. Polypeptide grafting was achieved via EDC / NHS activation (grafting rate 5.46%–6.50%). Experimental results show that the hydrogels possess excellent physicochemical properties, exhibiting long-term permeability stability (uniform phenol red diffusion), controllable mechanical strength (modulus 10–200 Pa), and slow degradation characteristics (11.6% decrease in sodium alginate viscosity after 7 days), with a stable pH of 7.2–7.4. Biocompatibility experiments indicate that the hydrogels are non-toxic to osteoblasts, nerve cells, and angiocytes, have a high proportion of viable cells, and can significantly promote cell proliferation, making them particularly suitable for long-term three-dimensional culture of osteoblasts and nerve cells. However, angiocytes are prone to disrupting the gel structure due to synaptic growth, requiring further cell experiments. Overall, these sodium alginate-based hydrogels show significant potential in tissue engineering and organoid culture, demonstrating promising applications in tissue engineering.

[0160] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A sodium alginate-based hydrogel, characterized in that, The hydrogel is a basic hydrogel, and its raw materials include the following components: low-acylated gellan gel powder, high-acylated gellan gel powder, RGD-SA powder, VN-SA powder, QK-SA powder, deionized water, and α-MEM culture medium. The RGD-SA powder, VN-SA powder, and QK-SA powder were prepared by freeze-drying of peptide-modified functionalized sodium alginate RGD-SA, VN-SA, and QK-SA solutions, respectively. The RGD-SA powder, VN-SA powder, and QK-SA powder were prepared by reacting RGD polypeptide, VN polypeptide, and QK polypeptide with sodium alginate in a molar ratio of 1:15, respectively. The raw material amounts for the basic hydrogel are as follows: 0.72g of low-acylated gellan gel powder, 0.08g of high-acylated gellan gel powder, 0.08g of RGD-SA powder, 0.10g of VN-SA powder, 0.02g of QK-SA powder, 100mL of deionized water, and 50mL of α-MEM culture medium.

2. A sodium alginate-based hydrogel, characterized in that, The hydrogel is an organoid hydrogel, and its raw materials include the following components: low-acylated gellan gel powder, high-acylated gellan gel powder, RGD-SA powder, VN-SA powder, QK-SA powder, deionized water, and α-MEM culture medium. The RGD-SA powder, VN-SA powder, and QK-SA powder were prepared by freeze-drying of peptide-modified functionalized sodium alginate RGD-SA, VN-SA, and QK-SA solutions, respectively. The RGD-SA powder, VN-SA powder, and QK-SA powder were prepared by reacting RGD polypeptide, VN polypeptide, and QK polypeptide with sodium alginate in a molar ratio of 1:15, respectively. The raw materials used in the organoid hydrogel are as follows: 0.72g of low-acylated gellan gel powder, 0.08g of high-acylated gellan gel powder, 0.02g of RGD-SA powder, 0.10g of VN-SA powder, 0.08g of QK-SA powder, 100mL of deionized water, and 50mL of α-MEM culture medium.

3. A method for preparing a sodium alginate-based hydrogel, wherein the sodium alginate-based hydrogel is the sodium alginate-based hydrogel according to any one of claims 1 and 2, characterized in that: Includes the following steps: S1: N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to morpholine ethanesulfonic acid buffer to dissolve and prepare an activation solution, which was then filtered to remove bacteria. S2: Dissolve RGD peptide, VN peptide and QK peptide in phosphate buffer to prepare peptide working solutions; S3: Add sodium alginate to phosphate buffer and stir to dissolve; S4: Add the activation solution to the dissolved sodium alginate solution and stir. Then add the peptide working solution and stir to obtain peptide-modified functionalized sodium alginate RGD-SA, VN-SA and QK-SA solutions. After freeze-drying, obtain RGD-SA powder, VN-SA powder and QK-SA powder. S5: Dissolve low-acylated gellan gum powder, high-acylated gellan gum powder, RGD-SA powder, VN-SA powder and QK-SA powder in deionized water in different proportions to obtain basic hydrogel uncrosslinked solution and organoid hydrogel uncrosslinked solution, and filter and sterilize them in a clean bench. S6: Mix the uncrosslinked solutions of the basic hydrogel and the organoid hydrogel with α-MEM culture medium in a certain proportion, and wait for the reaction to crosslink into gel to obtain the basic hydrogel and the organoid hydrogel.

4. The method for preparing a sodium alginate-based hydrogel according to claim 3, characterized in that: In step S1, a 0.1 mol / L morpholine ethanesulfonic acid buffer solution is prepared and the pH is adjusted to 6.0-7.

5. 0.06 g of N-hydroxysuccinimide and 0.2 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are added to 20 mL of morpholine ethanesulfonic acid buffer solution and fully dissolved to prepare the activation solution. The solution is then filtered through a 0.22 μm filter in a clean bench and set aside for use.

5. The method for preparing a sodium alginate-based hydrogel according to claim 4, characterized in that: In step S2, RGD peptide, VN peptide, and QK peptide are dissolved in 1 mL of phosphate buffer at a molar ratio of RGD:SA = 1:15, respectively, to prepare working solutions for the peptides.

6. The method for preparing a sodium alginate-based hydrogel according to claim 5, characterized in that: In step S3, 1g of sodium alginate is added to 50mL of phosphate buffer and magnetically stirred for 4h to fully dissolve the sodium alginate.

7. The method for preparing a sodium alginate-based hydrogel according to claim 6, characterized in that: In step S4, at room temperature, 175µL of EDC / NHS activation solution is added to 50mL of fully dissolved sodium alginate solution. After magnetic stirring for 1h, the solution is allowed to stand for 3h to allow the sodium alginate to activate. Then, 100µL of the prepared peptide working solution is added to the activated sodium alginate solution and magnetically stirred for 1h to obtain peptide-modified functionalized sodium alginate RGD-SA, VN-SA and QK-SA solutions. The solutions are then transferred to a 4℃ refrigerator and allowed to stand for 20h.

8. The method for preparing a sodium alginate-based hydrogel according to claim 7, characterized in that: In step S4, the mixed solution is dialyzed at 4°C for three days using a dialysis bag. The dialysate is changed every 6 hours on the first day, and every 12 hours on the second and third days. After dialysis, the solution is filtered to remove bacteria, and then aseptically freeze-dried to obtain RGD-SA powder, VN-SA powder and QK-SA powder.

9. The method for preparing a sodium alginate-based hydrogel according to claim 5, characterized in that: In step S5, 0.72g of low-acylated gellan gum powder, 0.08g of high-acylated gellan gum powder, 0.08g of RGD-SA powder, 0.10g of VN-SA powder and 0.02g of QK-SA powder are dissolved in 100mL of deionized water at 37°C to obtain a basic hydrogel uncrosslinked solution, which is then filtered and sterilized in a clean bench.

10. The method for preparing a sodium alginate-based hydrogel according to claim 5, characterized in that: In step S5, 0.72g of low-acylated gellan gum powder, 0.08g of high-acylated gellan gum powder, 0.02g of RGD-SA powder, 0.10g of VN-SA powder, and 0.08g of QK-SA powder are dissolved in 100mL of deionized water at 37°C to obtain an organoid hydrogel uncrosslinked solution, which is then filtered and sterilized in a clean bench. In step S6, the basic hydrogel uncrosslinked solution is mixed with α-MEM culture medium at a ratio of 2:1 and reacted for 5 minutes until crosslinking occurs, thus obtaining the basic hydrogel. The organoid hydrogel was obtained by mixing the uncrosslinked solution of the organoid hydrogel with α-MEM culture medium at a ratio of 2:1 and reacting for 5 minutes until crosslinking occurred.

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