A multifunctional fucoidan 3D printing hydrogel for tissue engineering, its preparation method and application

Abstract

This invention relates to a method for preparing and applying a multifunctional fucoidan 3D-printed hydrogel for tissue engineering, comprising the following steps: Step 1, dissolving fucoidan powder to obtain a fucoidan aqueous solution; Step 2, degrading the fucoidan aqueous solution under high temperature and high pressure; Step 3, dialysis, freezing, and drying the degradation solution obtained in Step 2 to obtain depolymerized fucoidan powder; Step 4, dissolving the depolymerized fucoidan powder to obtain a depolymerized fucoidan solution; Step 5, modifying the depolymerized fucoidan solution; Step 6, dialysis, freezing, and drying the chemically modified depolymerized fucoidan solution to obtain modified depolymerized fucoidan powder; Step 7, mixing the modified depolymerized fucoidan powder with a photoinitiator to obtain a hydrogel precursor solution; Step 8, photocrosslinking the hydrogel precursor solution to print a multifunctional fucoidan 3D-printed hydrogel for tissue engineering.

Description

Technical Field

[0001] This invention relates to the field of new biomaterials, particularly to the development of 3D printing hydrogels and 3D printing bio-ink materials, specifically to a multifunctional fucoidan 3D printing hydrogel for tissue engineering, its preparation method, and its applications. Background Technology

[0002] With the rapid development of tissue engineering and regenerative medicine, researchers have been exploring new treatment methods for tissue and organ regeneration and replacement in recent years. Biomaterials (naturally extracted or synthesized) have shown great potential in regeneration and repair, and have become suitable alternatives for tissues and organs. 3D bioprinting technology, incorporating biomaterials, has attracted significant attention and has become an important tool for advancing tissue engineering and regenerative medicine. Through integrated manufacturing processes, this technology can precisely deposit biomaterials and cells into tissue or organ models in a controlled manner, thereby generating personalized living tissue structures with complex geometries—something difficult to achieve with traditional biomanufacturing methods. Currently, this technology is widely used in clinical transplant tissue manufacturing, disease pathology modeling, and drug compound screening, demonstrating outstanding therapeutic potential in tissue engineering and regenerative medicine. The key to bioprinting technology lies in the selection of suitable biomaterials, which provide a stable, biocompatible, and biomimetic extracellular matrix space for cell proliferation and differentiation. However, the limited selection of biomaterials currently available for 3D bioprinting severely restricts its application in tissue engineering. Currently available synthetic materials for 3D bioprinting, such as PEG-DA and poly(caprolactone) (PCL), cause inflammatory reactions due to poor biocompatibility. Available natural materials are limited to alginate, gelatin, collagen, and silk fibroin. However, the biological functions of most of these natural biomaterials still cannot satisfactorily address a series of problems frequently encountered in tissue and organ regeneration, such as bacterial infection, excessive reactive oxygen species (ROS), and external pressure. Therefore, there is an urgent need to develop multifunctional biomaterials that simultaneously possess excellent biocompatibility, good antibacterial and antioxidant properties, and outstanding mechanical properties to meet the needs of bioprinting technology development and its application in tissue engineering.

[0003] Prior art 1: Chinese invention patent, entitled "A Highly Effective Antibacterial Hydrogel Wound Dressing for Promoting Wound Healing and Its Preparation Method" (application number: 2022104523027), discloses a dressing mainly composed of polyaspartic acid derivative (PPDB) and polyvinyl alcohol (PVA), used for the treatment of bacterial infected wounds. Its antibacterial effect mainly originates from the photothermal effect of doped small molecule peptide-modified dopamine nanoparticles and the cationic quaternary ammonium salt groups carried by the polyaspartic acid derivative (PPDB). However, the incorporation of these antibacterial agents inevitably produces a certain degree of cytotoxicity.

[0004] Prior art 2: Chinese invention patent, entitled "An antibacterial and antioxidant hydrogel dressing for treating diabetic wounds and its preparation method" (application number: 2022105998409), discloses a method of crosslinking an epoxy-containing gelling backbone with an amino-containing hyperbranched polylysine-manganese dioxide nanosheet composite to prepare a multifunctional injectable hydrogel with antibacterial and reactive oxygen species scavenging properties. However, the inherent immune rejection of manganese dioxide nanosheets can affect the health of the recipient to some extent.

[0005] Prior art 3: Chinese invention patent, entitled: A 3D printing high-strength bio-ink material (application number: 2017113484054), discloses a 3D printing high-strength bio-ink constructed from a double cross-linked network structure, using silk protein that forms a β-sheet structure and ion-crosslinked alginate. This bio-ink is mainly composed of a mixture of well-known commercially available bio-ink materials.

[0006] None of the aforementioned existing technologies have achieved the development of a multifunctional, single, all-natural biomaterial that simultaneously possesses excellent biocompatibility, good antibacterial and antioxidant properties, and outstanding mechanical properties. Summary of the Invention

[0007] This invention designs a multifunctional fucoidan 3D printing hydrogel for tissue engineering, its preparation method, and its application. The technical problem it solves is that the biological functions of existing natural biomaterials still cannot satisfactorily address the problems frequently encountered in tissue and organ regeneration, such as bacterial infection, overexpressed reactive oxygen species (ROS), and external pressure.

[0008] To solve the aforementioned technical problems, the present invention adopts the following solution:

[0009] A method for preparing a multifunctional fucoidan 3D printing hydrogel for tissue engineering includes the following steps:

[0010] Step 1: Dissolve the fucoidan powder to obtain a fucoidan aqueous solution;

[0011] Step 2: High-temperature and high-pressure degradation of fucoidan aqueous solution;

[0012] Step 3: The degradation solution obtained in Step 2 is dialyzed, frozen, and dried to obtain depolymerized fucoidan powder;

[0013] Step 4: Dissolve the depolymerized fucoidan powder to obtain a depolymerized fucoidan solution;

[0014] Step 5: The depolymerized fucoidan solution was modified with glycidyl methacrylate;

[0015] Step 6: Dialyze, freeze-dry, and then dialyze the chemically modified depolymerized fucoidan solution to obtain modified depolymerized fucoidan powder.

[0016] Step 7: The modified depolymerized fucoidan powder is mixed with a photoinitiator to obtain a hydrogel precursor solution;

[0017] Step 8: Photocrosslinking of hydrogel precursor solution to print multifunctional tissue engineering fucoidan 3D printed hydrogels.

[0018] Preferably, in step 1, the fucoidan powder is dissolved in deionized water and heated and stirred at 40-90°C until completely dissolved.

[0019] Preferably, in step 2, the temperature is controlled at 100-130℃ and the pressure is controlled at 0.1-0.2MPa.

[0020] Preferably, the hydrogel precursor solution in step 7 is a modified depolymerized fucoidan hydrogel precursor solution with a concentration of 1-5%wt.

[0021] Preferably, in step 8, the modified depolymerized fucoidan hydrogel precursor solution is added to the material tank of a 3D printing device with 405nm wavelength light-assisted printing, and printing is performed according to a preset digital model and program. The light irradiation time is 6s / 100μm, and the light intensity is 15mW / cm². 2 .

[0022] The above preparation method yields a multifunctional fucoidan 3D printing hydrogel for tissue engineering.

[0023] The multifunctional fucoidan 3D printing hydrogel for tissue engineering of this invention can be applied to cartilage defect repair or tissue wound repair, but is not limited to the above uses.

[0024] A method for preparing a hydrogel precursor solution includes the following steps: Step 1, fucoidan degradation; Step 2, modification of the degradation product obtained in Step 1; Step 3, mixing the modified product obtained in Step 2 with a photoinitiator to obtain the hydrogel precursor solution.

[0025] The multifunctional fucoidan 3D printing hydrogel for tissue engineering, its preparation method, and its application have the following beneficial effects:

[0026] (1) This invention uses depolymerized fucoidan obtained through controlled degradation as the main raw material. Through simple methacrylation, a multifunctional hydrogel biomaterial with biocompatibility, antibacterial, antioxidant and high strength that can be 3D printed is obtained. It has advantages that are not available in currently known single natural biomaterials.

[0027] (2) This invention discovers the antibacterial, antioxidant and other biological activities of fucoidan through controlled degradation. Compared with other technologies for developing multifunctional biomaterials, it avoids the doping of other potentially toxic reagents or drugs, and exerts its excellent biological activity while maintaining its unique single natural component. Attached Figure Description

[0028] Figure 1 This is a flowchart illustrating the preparation process of the multifunctional fucoidan 3D printing hydrogel for tissue engineering according to the present invention.

[0029] Figure 2 : Schematic diagram of chemical modification of depolymerized fucoidan solution in this invention;

[0030] Figure 3 : Comparison of NMR spectra of fucoidan solution before and after depolymerization in this invention;

[0031] Figure 4 A comparison diagram of the gel model printed in this invention and the CAD design model;

[0032] Figure 5 : A diagram showing the growth state of chondrocytes in hydrogel in this invention;

[0033] Figure 6 Comparison chart of mechanical strength test results of this invention;

[0034] Figure 7 A comparison chart of colony counts and antibacterial rates calculated on agar plates in this invention;

[0035] Figure 8 Comparison of ultraviolet absorption spectra and DPPH scavenging rates in this invention; Detailed Implementation

[0036] The following is combined with Figure 1-8 The present invention will be further described as follows:

[0037] like Figure 1As shown, the preparation method of the multifunctional fucoidan 3D printing hydrogel for tissue engineering of the present invention includes the following steps: Step 1, dissolving fucoidan powder to obtain fucoidan aqueous solution; Step 2, degrading the fucoidan aqueous solution under high temperature and high pressure; Step 3, dialysis, freezing, and drying the degradation solution obtained in Step 2 to obtain depolymerized fucoidan powder; Step 4, dissolving the depolymerized fucoidan powder to obtain depolymerized fucoidan solution; Step 5, modifying the depolymerized fucoidan solution with glycidyl methacrylate; Step 6, dialysis, freezing, and drying the chemically modified depolymerized fucoidan solution to obtain modified depolymerized fucoidan powder; Step 7, mixing and dissolving the modified depolymerized fucoidan powder with lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) to obtain a hydrogel precursor solution; Step 8, photocrosslinking the hydrogel precursor solution to print a multifunctional fucoidan 3D printing hydrogel for tissue engineering. Example

[0038] 1) Weighing and dissolving:

[0039] Weigh 3g of fucoidan powder, place it in a beaker, add 100 mL of deionized water, heat and stir at 50 ℃ (500 rpm) for 2 h until the raw material is completely dissolved.

[0040] 2) High temperature and high pressure degradation:

[0041] The obtained 3% fucoidan aqueous solution was placed in a high-pressure reactor for degradation. The temperature was set at 121 °C, the pressure at 0.13 MPa, and the degradation time at 40 min.

[0042] 3) Dialysis, freeze-drying:

[0043] The depolymerized fucoidan solution obtained after degradation was dialyzed in deionized water for 3 days. The dialysis bag used had a molecular weight cutoff of 500 DP, and the deionized water used for dialysis was changed three times a day during this period. After 3 days, the dialysis solution was freeze-dried to obtain depolymerized fucoidan powder.

[0044] 4) Weigh and dissolve:

[0045] Weigh 1g of the obtained depolymerized fucoidan powder, place it in a beaker, add 100 mL of deionized water, heat and stir at 50 ℃ (200 rpm) for 0.5 h until the raw material is completely dissolved.

[0046] 5) Chemical modification:

[0047] Glycidyl methacrylate (0.6 g by mass) was added dropwise to the obtained depolymerized fucoidan solution, and the mixture was magnetically stirred at room temperature in the dark for 8 hours. The chemical modification results are as follows: Figure 2As shown, this indicates that the hydroxyl groups in the depolymerized fucoidan are replaced and grafted with carbon-carbon double bond groups. The NMR comparison images before and after chemical modification are shown below. Figure 3 As shown, the results indicate successful grafting of carbon-carbon double bonds and methyl groups, and the three chemical modification degrees of depolymerized fucoidan were calculated to be 35.4%, 38.8%, and 42.3% respectively, based on the NMR peak integrated area.

[0048] 6) Dialysis, freeze-drying:

[0049] The chemically modified depolymerized fucoidan solution was dialyzed in deionized water for 7 days. The dialysis bag used had a molecular weight cutoff of 200 DP, and the deionized water was changed three times a day during the dialysis period. After 7 days, the dialysis solution was freeze-dried to obtain modified depolymerized fucoidan powder.

[0050] 7) Weigh and dissolve:

[0051] Weigh 0.2 g of the obtained modified depolymerized fucoidan powder and 5 mg of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) into a 10 mL centrifuge tube, add 10 mL of deionized water, and dissolve all the raw materials to obtain a hydrogel precursor solution with a modified depolymerized fucoidan concentration of 2%wt.

[0052] 8) Photocrosslinking printing:

[0053] The hydrogel precursor solution was added to the material tank of a 3D printing device assisted by 405nm wavelength light, and printing was performed according to a preset digital model and program. The light exposure time was 6s / 100μm, and the light intensity was 15mW / cm². 2 Thus, the 3D-printed hydrogel based on fucoidan of this invention is obtained. The comparison results between the printed gel model and the CAD design model are as follows: Figure 4 As shown, the printed hydrogel can perfectly reproduce the shape and size of the design model, has excellent 3D printing fidelity, and the cells encapsulated in the gel model are in good condition.

[0054] 9) Biocompatibility, antibacterial and antioxidant tests:

[0055] The biocompatibility of the material was tested using the FDA staining method: 5*10⁻⁶ ppm was added to the hydrogel precursor solution. 6 / mL of live chondrocytes were mixed thoroughly and then transferred into a polytetrafluoroethylene mold (φ10mm×2mm). The mold was then irradiated at a wavelength of 405nm for 30s with a light intensity of 15 mW / cm². 2 Thus, the bio-manufactured hydrogel for tissue repair based on fucoidan of the present invention is obtained.

[0056] Subsequently, fluorescein diacetate was used to stain the live cells in the hydrogel to observe the growth status of chondrocytes in the hydrogel on day 7. Figure 5 The results showed that chondrocytes were evenly distributed in the three-dimensional structure of the hydrogel, appearing as spherical cells and partially agglomerated, maintaining the growth state of chondrocytes in vivo. Furthermore, the fluorescence results on day 7 were significantly enhanced compared to day 1, indicating that the hydrogel material can effectively promote cell proliferation.

[0057] The mechanical strength of the material was tested using a universal testing machine: 100 μL of hydrogel precursor solution was transferred into a polytetrafluoroethylene mold (φ10 mm × 2 mm), and irradiated at a wavelength of 405 nm for 30 s with an irradiation intensity of 15 mW / cm². 2 Prepare the hydrogel required for the test. Place the hydrogel on the testing machine, which has a strain gauge of 20 N and an accuracy of 0.0001 N. The machine begins compression at a rate of 1 mm / min, with a displacement accuracy of 0.0001 mm. Record the compressive force until the hydrogel structure breaks down. The mechanical strength test results are as follows: Figure 6 As shown, the results indicate that the hydrogel has a compressive modulus of up to 311 kPa, which is 14.8 times that of the current gold standard product GelMA.

[0058] The antibacterial properties of the material were tested using the plate coating method: 100 μL of hydrogel precursor solution was transferred into a polytetrafluoroethylene mold (φ10 mm × 2 mm), and irradiated at a wavelength of 405 nm for 30 s with an irradiation intensity of 15 mW / cm². 2 Hydrogels were prepared and placed in 48-well plates. Then, bacterial suspensions (400 μL, 2.5 × 10⁻⁶ μL) were added. 4 Add cfu / mL to the wells of the plate and incubate with the hydrogel. Use 400 μL of pure bacteria as a control, without hydrogel. After incubation at 37°C for 2 hours, remove 200 μL of bacterial solution from the wells and spread it evenly on an agar plate (for Escherichia coli) or a blood plate (for Staphylococcus aureus). After 24 hours of incubation, count the colonies on the plates and calculate the antibacterial rate. Figure 7 As shown, the results indicate that the number of bacterial colonies in the experimental group was significantly lower than that in the pure bacteria control group. The antibacterial rates against Escherichia coli and Staphylococcus aureus, as calculated by Imagej software, were 94.2% and 97.5%, respectively.

[0059] The antioxidant properties of the material were tested using the DPPH method: 100 μL of hydrogel precursor solution was transferred into a polytetrafluoroethylene mold (φ10 mm × 2 mm), and irradiated at a wavelength of 405 nm for 30 s with an irradiation intensity of 15 mW / cm². 2 Hydrogels were prepared and in ethanol solution of DPPH (2×10⁻⁶) -4The free radical scavenging activity was measured by incubating the product in mol / L solution under dark conditions for 4 hours, and then detecting the absorbance at 517 nm using a UV-Vis spectrometer. The DPPH free radical scavenging activity was calculated using the following formula:

[0060] DPPH removal rate = (Ac-As) / Ac × 100%;

[0061] Where Ac refers to the absorbance of the pure DPPH ethanol solution, and As refers to the absorbance of the DPPH solution in which the hydrogel is cultured. The UV absorption spectrum and DPPH scavenging rate are shown below. Figure 8 As shown in the figure. The results showed that, compared with the control group and the GelMA group, the peak value of the characteristic absorption peak of DPPH (517 nm) in the experimental group was significantly reduced. Quantitative analysis showed that the DPPH scavenging rate of the experimental group reached 63.1%.

[0062] The present invention has been described above by way of example with reference to the accompanying drawings. Obviously, the implementation of the present invention is not limited to the above-described manner. Any improvements made using the inventive concept and technical solution of the present invention, or the direct application of the inventive concept and technical solution of the present invention to other occasions without modification, are all within the protection scope of the present invention.

Claims

1. A method for preparing a multifunctional fucoidan 3D printing hydrogel for tissue engineering, comprising the following steps: Step 1: Dissolve the fucoidan powder to obtain a fucoidan aqueous solution; Step 2: High-temperature and high-pressure degradation of fucoidan aqueous solution; in Step 2, the temperature is controlled at 100-130℃ and the pressure is controlled at 0.1-0.2MPa. Step 3: The degradation solution obtained in Step 2 is dialyzed, frozen, and dried to obtain depolymerized fucoidan powder; Step 4: Dissolve the depolymerized fucoidan powder to obtain a depolymerized fucoidan solution; Step 5: Modification of depolymerized fucoidan solution; in step 5, glycidyl methacrylate or methacrylic anhydride is used for modification; Step 6: Dialyze, freeze-dry, and then dialyze the chemically modified depolymerized fucoidan solution to obtain modified depolymerized fucoidan powder. Step 7: The modified depolymerized fucoidan powder is mixed with a photoinitiator to obtain a hydrogel precursor solution; the photoinitiator in Step 7 is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) or 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (I2959); the hydrogel precursor solution in Step 7 is a hydrogel precursor solution with a modified depolymerized fucoidan concentration of 1-5%wt. Step 8: Photocrosslinking of hydrogel precursor solution to print multifunctional tissue engineering fucoidan 3D printed hydrogels.

2. The method for preparing the multifunctional fucoidan 3D printing hydrogel for tissue engineering according to claim 1, characterized in that: In step 1, the fucoidan powder is dissolved in deionized water and heated and stirred at 40-90℃ until completely dissolved.

3. The method for preparing the multifunctional fucoidan 3D printing hydrogel for tissue engineering according to claim 1, characterized in that: In step 8, the modified depolymerized fucoidan hydrogel precursor solution is added to the material tank of a 3D printing device with 405nm wavelength light-assisted printing. Printing is performed according to the preset digital model and program, with an illumination duration of 6s / 100μm and an illumination intensity of 15mW / cm². 2 .

4. A multifunctional fucoidan 3D printing hydrogel for tissue engineering prepared by the preparation method according to any one of claims 1-3.

5. The multifunctional tissue engineering fucoidan 3D printing hydrogel of claim 4 is used to prepare materials for cartilage defect repair or tissue wound repair.

6. A method for preparing a hydrogel precursor solution, comprising the following steps: Step 1: Degradation of fucoidan; temperature controlled at 100-130℃, pressure controlled at 0.1-0.2MPa; Step 2: Modification of the degradation product obtained in Step 1; Step 3: The modified product obtained in Step 2 is mixed with a photoinitiator to obtain a hydrogel precursor solution; The modification was performed using glycidyl methacrylate or methacrylic anhydride; the photoinitiator was lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) or 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (I2959); the hydrogel precursor solution was a modified depolymerized fucoidan hydrogel precursor solution with a concentration of 1-5%wt.

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