Preparation method of blood-derived GeLMA-lignin bionic hydrogel scaffold

Abstract

The invention belongs to the technical field of biomedical materials and bone tissue engineering, and particularly relates to a preparation method of a blood-derived GeLMA-lignin bionic hydrogel scaffold. Comprising the following steps: S1, preparing a GeLMA hydrogel precursor; s2, preparing lignin nano particles; s3, preparing PRF powder; s4, preparing the LIG / PRF nano particles; s5, preparing the blood-derived GeLMA-lignin bionic hydrogel scaffold. The single cold source ice template technology can control the pore direction, and the structure is highly similar to cancellous bone; triple functions of mechanical enhancement, anti-oxidation protection and growth factor slow release are realized through lignin nanoparticles; pRF is derived from autoblood and has the optimal physiological proportion and synergistic effect, and proportional imbalance and immunological rejection possibly caused by addition of exogenous factors are avoided; and a single cold source process structure is high in controllability and has industrialization potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and bone tissue engineering technology, specifically to a method for preparing a blood-derived GeLMA-lignin biomimetic hydrogel scaffold. Background Technology

[0002] Autologous bone grafting is still widely used in clinical bone defect repair, but this method has several limitations: (1) it requires opening a second bone harvesting site, increasing patient trauma and surgical risks; (2) the amount of bone harvested is limited, and a second surgery is often required to remove the fixation device; (3) allogeneic or artificial bone grafting has problems such as immune rejection and low osteogenic efficiency. Therefore, developing a novel bone tissue engineering scaffold with good biocompatibility, biodegradability and absorption, and the ability to promote osteogenic formation is of great clinical significance.

[0003] In recent years, GeLMA (a hydrogel-like material) has been widely used in the construction of tissue engineering scaffolds due to its excellent biocompatibility and cell adhesion. However, the main drawbacks of GeLMA hydrogels are insufficient mechanical strength, poor antioxidant properties, and limited bioactivity, which restricts their application in weight-bearing bone repair. Furthermore, in clinical practice, elevated levels of reactive oxygen species (ROS) during defect healing can lead to oxidative stress and cell damage. Oxidative stress inhibits mesenchymal cell osteogenic differentiation by suppressing the Hedgehog signaling pathway and can also antagonize the Wnt signaling pathway in osteoblast precursors by transferring β-Catenin to FoxO-mediated transcription, significantly impacting the final osteogenic outcome. Therefore, improving the mechanical properties of GeLMA and endowing it with antioxidant and angiogenesis-promoting capabilities to synergistically promote osteogenic development remains a key research challenge.

[0004] To address these issues, we provide a method for preparing a blood-derived GeLMA-lignin biomimetic hydrogel scaffold. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] To address the aforementioned technical problems, according to one aspect of the present invention, the present invention provides the following technical solution:

[0007] A method for preparing a blood-derived GeLMA-lignin biomimetic hydrogel scaffold includes the following steps:

[0008] S1: Preparation of GeLMA hydrogel precursor: Add 5g of porcine gelatin to PBS, heat in a water bath, stir and dissolve, add methacrylic anhydride, stir to obtain GeLMA hydrogel precursor solution, adjust pH=7, dialyze, and freeze dry.

[0009] S2: Preparation of lignin nanoparticles: Add alkaline lignin to methanol, stir until fully dissolved, centrifuge to remove insoluble impurities at the bottom, add deionized water, stir, centrifuge to collect the precipitate, and freeze-dry.

[0010] S3: Preparation of PRF powder: PRF powder was prepared by taking arterial blood from SD rats;

[0011] S4: Preparation of LIG / PRF nanoparticles;

[0012] S5: Preparation of blood-derived GeLMA-lignin biomimetic hydrogel scaffold:

[0013] S5.1: Weigh 120 mg of GeLMA hydrogel precursor and 1 mg of LIG / PRF nanoparticles, dissolve them in 1 ml of PBS solution containing 100 μl of LAP, protect from light, in a 37°C water bath, and shake until fully dissolved;

[0014] S5.2: Transfer the solution into the copper molds respectively. After the ice mold machine is fully pre-cooled to 45°C, place the molds on the cold source of the ice mold machine. As the ice crystals crystallize, they are irradiated with ultraviolet light and photocured. After the solution is completely frozen, continue to freeze in the ice mold for 2 hours.

[0015] S5.3: Take the sample, freeze it in liquid nitrogen for 10 seconds, then incubate it at -80℃ overnight, freeze-dry it for 24 hours, demold it, and obtain the blood-derived GeLMA-lignin biomimetic hydrogel scaffold.

[0016] In a preferred embodiment of the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold described in this invention, the methacrylic anhydride in S1 is added slowly and dropwise at a rate of 0.3 ml / min, with a total addition volume of 6 ml.

[0017] As a preferred embodiment of the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold described in this invention, the dialysis method in S1 is as follows: the pH-adjusted GeLMA hydrogel precursor solution is transferred to a dialysis bag with a molecular weight cutoff of 8000 D-14000 D, and dialyzed in deionized water at 40°C for seven days, during which the deionized water is replaced every three hours.

[0018] As a preferred embodiment of the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present invention, the specific method of S3 is as follows: S3.1: Take 5ml of arterial blood from SD rats into a centrifuge tube without anticoagulant and centrifuge at 3000rpm for 12 minutes.

[0019] S3.2: After centrifugation, the layers are separated. The upper layer is taken, placed between two gauze sheets, and pressed to remove water to obtain a PRF membrane.

[0020] S3.3: After freeze-drying the PRF membrane for 24 hours, grind it thoroughly to obtain PRF powder.

[0021] As a preferred embodiment of the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present invention, the specific method of S4 is as follows: S4.1: 10 mg of lignin nanoparticles are resuspended in 100 μl of PBS;

[0022] S4.2: Then, 3 mg of PRF powder was added to the resuspended lignin nanoparticle solution, fully dissolved, and incubated at 37°C for 24 hours;

[0023] S4.3: The sample was placed at -80℃ overnight, and then freeze-dried for 24 hours to obtain LIG / PRF nanoparticles.

[0024] Compared with existing technologies, the beneficial effects of this invention are: 1. Enhanced biomimicry: The single-source cold-source ice template technology can control the pore direction, resulting in a structure highly similar to cancellous bone; 2. Multifunctional integrated design: Lignin nanoparticles achieve a triple function of mechanical reinforcement, antioxidant protection, and sustained release of growth factors; 3. Advantages of endogenous growth factors: PRF is derived from autologous blood, possessing the optimal physiological ratio and synergistic effect, avoiding the imbalance and immune rejection that may be caused by the addition of exogenous factors; 4. Biodegradable and requiring no secondary surgery: The scaffold gradually degrades and is absorbed in the body, eliminating the need for secondary removal and reducing patient suffering; 5. Simple preparation and scalable: The single-source cold-source process offers strong structural controllability and has industrialization potential. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and detailed embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0026] Figure 1 This is a flowchart illustrating the preparation method of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold according to the present invention;

[0027] Figure 2 This is a TEM image of lignin nanoparticles (LIG NP) used in the preparation method of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold according to the present invention.

[0028] Figure 3 LIG / PRFTEM image of a method for preparing a blood-derived GeLMA-lignin biomimetic hydrogel scaffold according to the present invention;

[0029] Figure 4 The image shows the synthesized GeLMA-lignin biomimetic hydrogel scaffold according to the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present invention.

[0030] Figure 5 This is a microstructure diagram of the scaffold used in the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present invention.

[0031] Figure 6 This is a 10x SEM image of the cross-section of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold, as described in the present invention.

[0032] Figure 7 This is a 200x SEM image of the cross-section of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold, according to the present invention.

[0033] Figure 8 This invention relates to a method for preparing a blood-derived GeLMA-lignin biomimetic hydrogel scaffold, specifically the Young's modulus of GeLMA / LNP / PRF containing concentrations of 0.8 mg / ml LIG / PRF NP, 1 mg / ml LIG / PRF NP, and 1.2 mg / ml LIG / PRF NP (L: 0.8 mg / ml LIG / PRF nanoparticles added to the scaffold; M: 1 mg / ml LIG / PRF nanoparticles added to the scaffold; H: 1.2 mg / ml LIG / PRF nanoparticles added to the scaffold).

[0034] Figure 9 Fourier transform infrared spectrum of GeLMAGeLMA / LNP GeLMA / LNP / PRF scaffold, which is a method for preparing a blood-derived GeLMA-lignin biomimetic hydrogel scaffold according to the present invention.

[0035] Figure 10This is a CCK-8 biocompatibility assay of GeLMA / LNP / PRF containing concentrations of 0.8 mg / ml LIG / PRF NP, 1 mg / ml LIG / PRF NP, and 1.2 mg / ml LIG / PRF NP in a method for preparing a blood-derived GeLMA-lignin biomimetic hydrogel scaffold according to the present invention (L: 0.8 mg / ml LIG / PRF nanoparticles added to the scaffold; M: 1 mg / ml LIG / PRF nanoparticles added to the scaffold; H: 1.2 mg / ml LIG / PRF nanoparticles added to the scaffold).

[0036] Figure 11 The metabolic activity of each scaffold (CON, blank group, GeL, simple GeL scaffold, GEL / LNP, and GEL / LNP / PRF, lignin nanoparticles loaded with PRF growth factor added to GeLMA hydrogel) on BMSCs was detected by CCK-8 assay.

[0037] Figure 12 The image shows the staining process observed under a confocal microscope after co-culturing the material extract with BMSCs for 1 day, 4 days, and 7 days, as part of the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present invention.

[0038] Figure 13 This is a confocal microscope image showing the live / dead staining after bone marrow mesenchymal stem cells were seeded onto the scaffold at 1, 4, and 7 days, as part of the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present invention.

[0039] Figure 14 This is a fluorescence staining image of the in vivo ROS (reactive oxygen species) level in bmsc (bone marrow mesenchymal stem cells) in a method for preparing a blood-derived GeLMA-lignin biomimetic hydrogel scaffold according to the present invention.

[0040] Figure 15 This invention relates to a method for preparing a blood-derived GeLMA-lignin biomimetic hydrogel scaffold. Figure 14 Bar chart for quantitative analysis of fluorescence intensity;

[0041] Figure 16 The figure shows the Huvecs Transwell experimental results of GEL, GEL / LNP, and GEL / LNP / PRF scaffolds in the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present invention.

[0042] Figure 17The figure shows the tube formation results of GEL, GEL / LNP and GEL / LNP / PRF scaffolds in the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present invention.

[0043] Figure 18 The image shows the scratch test results of the huvecs of GEL, GEL / LNP, and GEL / LNP / PRF scaffolds in the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present invention.

[0044] Figure 19 This is an immunofluorescence image showing the protein expression of VEGF cytokine generated in cells and angiogenesis after co-culturing cells and materials in the preparation method of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present invention.

[0045] Figure 20 This is an immunofluorescence image showing the protein expression of HIF-1α cytokine generated in cells and angiogenesis after co-culturing cells and materials in the preparation method of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present invention.

[0046] Figure 21 The expression levels of mRNA of the angiogenesis VEGF cytokine in the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present invention are shown.

[0047] Figure 22 The expression levels of HIF-1α cytokine mRNA in relation to the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of this invention.

[0048] Figure 23 Early alkaline phosphatase staining images of GEL, GEL / LNP, and GEL / LNP / PRF scaffolds in the preparation method of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold according to the present invention.

[0049] Figure 24 Late alizarin red staining images of GEL, GEL / LNP, and GEL / LNP / PRF scaffolds in the preparation method of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold according to the present invention.

[0050] Figure 25 This is an immunofluorescence image showing the protein expression of osteogenic-related RUNX-2 cytokine in cells after co-culturing cells and materials in the preparation method of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present invention.

[0051] Figure 26This is an immunofluorescence image showing the protein expression of osteogenic-related OCN cytokines in cells after co-culturing cells and materials in the preparation method of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present invention.

[0052] Figure 27 The expression level of RUNX-2 cytokine mRNA related to bone formation is shown in the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present invention.

[0053] Figure 28 The expression levels of OCN cytokines related to bone formation are measured in the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present invention. Detailed Implementation

[0054] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0055] Secondly, the present invention is described in detail with reference to the schematic diagrams. When detailing the embodiments of the present invention, for ease of explanation, the cross-sectional views illustrating the device structure may be partially enlarged, not according to the usual scale. Furthermore, the schematic diagrams are merely examples and should not limit the scope of protection of the present invention. In addition, actual fabrication should include three-dimensional spatial dimensions of length, width, and depth.

[0056] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0057] This invention introduces lignin nanoparticles loaded with growth factors into the GeLMA hydrogel system, combined with single-source cold-template technology, to construct a bone tissue engineering scaffold with an anisotropic, porous biomimetic structure (similar to cancellous bone structure), achieving a unity of "structural biomimicry" and "functional biomimicry." The scaffold incorporates endogenous growth factor groups derived from platelet-rich plasma (PRF). These factors, originating from the patient's own blood, possess optimal physiological ratios and natural synergistic effects, significantly promoting osteoblast adhesion, proliferation, and differentiation, while avoiding the immune rejection and imbalance problems that may arise from heterologous growth factors. The lignin nanoparticles further endow the material with excellent mechanical properties and antioxidant functions, enabling the scaffold to possess three functions: mechanical strengthening, antioxidant properties, and bioactivity regulation.

[0058] This scaffold exhibits multiple mechanisms: 1. Mechanical reinforcement mechanism: Lignin nanoparticles form hydrogen bonds or covalent crosslinks with GeLMA molecular chains, increasing crosslink density and elastic modulus, thereby significantly enhancing the scaffold's compressive strength and deformation recovery performance. 2. Antioxidant mechanism: Lignin is rich in polyphenolic hydroxyl structures, which can effectively scavenge reactive oxygen species (ROS) generated during bone defect healing, alleviate cell damage caused by oxidative stress, maintain the stability of the osteogenic microenvironment, and promote bone repair. 3. Endogenous growth factor sustained-release mechanism: The PRP loaded in the scaffold originates from the patient's own blood and contains multiple growth factors such as PDGF, TGF-β, VEGF, and IGF, which coexist in optimal physiological proportions, exhibiting a natural synergistic promoting effect.

[0059] Lignin nanoparticles can serve as sustained-release carriers to achieve the controlled release of these endogenous growth factors, avoiding the explosive release that occurs when PRF is directly incorporated in traditional methods.

[0060] In this way, growth factors can be released stably and rhythmically within the material over a long period, promoting angiogenesis and new bone formation.

[0061] Specifically, a method for preparing a blood-derived GeLMA-lignin biomimetic hydrogel scaffold includes the following steps:

[0062] S1: Preparation of GeLMA hydrogel precursor:

[0063] S1.1: Heat 50ml of PBS in a water bath to 50℃, add 5g of pigskin gelatin, heat in a water bath at 50℃ and stir continuously at 500rpm for 2 hours until completely dissolved;

[0064] S1.2: 6 ml of methacrylic anhydride was slowly and dropwise added to the gelatin solution at a rate of 0.3 ml / min, followed by stirring at 500 rpm for 3 hours until the substitution reaction was completed, thus obtaining the GeLMA hydrogel precursor solution.

[0065] S1.3: Add NaOH solution dropwise to the GeLMA hydrogel precursor solution in small amounts several times, and measure the pH of the solution. When the pH is adjusted to 7, terminate the reaction.

[0066] S1.4: Transfer the pH-adjusted GeLMA hydrogel precursor solution into a dialysis bag with a molecular weight cutoff of 8000 D-14000 D, and dialyze it in deionized water at 40℃ for seven days. During this period, change the deionized water every three hours.

[0067] S1.5: After dialysis, the product was transferred to a glass and frozen at -80°C overnight. Then it was freeze-dried for 48 hours to obtain a porous foam-like GeLMA hydrogel precursor.

[0068] S2: Preparation of lignin nanoparticles (LIG NP):

[0069] S2.1: Add 60 mg of alkaline lignin to 10 ml of methanol and stir magnetically for 2 hours until fully dissolved;

[0070] S2.2: Transfer the solution to a centrifuge tube, centrifuge briefly at 800 rpm for 2 minutes, and remove insoluble impurities at the bottom;

[0071] S2.3: Transfer the solution to a beaker and slowly add deionized water to the solution while stirring magnetically. Add 6 ml of deionized water to every 2 ml of lignin solution. After adding all the water, continue stirring for 10 minutes.

[0072] S2.4: After stirring, take 10000×g of sample and centrifuge for 10 minutes. Collect the precipitate, incubate overnight at -80℃, and then freeze-dry for 24 hours to obtain lignin nanoparticles.

[0073] S3: Preparation of PRF powder:

[0074] S3.1: Take 5 ml of arterial blood from SD rats into a centrifuge tube without anticoagulant and centrifuge at 3000 rpm for 12 minutes;

[0075] S3.2: After centrifugation, the layers are separated. The upper layer is taken, placed between two gauze sheets, and pressed to remove water to obtain a PRF membrane.

[0076] S3.3: After freeze-drying the PRF membrane for 24 hours, grind it thoroughly to obtain PRF powder;

[0077] S4: Preparation of LIG / PRF nanoparticles:

[0078] S4.1: Resuspend 10 mg of lignin nanoparticles (LIG NP) in 100 μl of PBS;

[0079] S4.2: Then, 3 mg of PRF powder was added to the resuspended lignin nanoparticle solution, fully dissolved, and incubated at 37°C for 24 hours;

[0080] S4.3: The sample was placed at -80℃ overnight, and then freeze-dried for 24 hours to obtain LIG / PRF nanoparticles;

[0081] S5: Preparation of blood-derived GeLMA-lignin biomimetic hydrogel scaffold:

[0082] S5.1: Weigh 120 mg of GeLMA hydrogel precursor and 1 mg of LIG / PRF nanoparticles, dissolve them in 1 ml of PBS solution containing 100 μl of LAP, protect from light, in a 37°C water bath, and shake until fully dissolved;

[0083] S5.2: Transfer the solution into the copper molds respectively. After the ice template machine is fully pre-cooled to 45°C, place the molds on the cold source of the ice template machine. While the ice crystals are crystallizing, irradiate with ultraviolet light (405nm) for photocuring. After the solution is completely frozen, continue to freeze in the ice template for 2 hours.

[0084] S5.3: Take the sample, freeze it in liquid nitrogen for 10 seconds, then incubate it at -80℃ overnight, freeze-dry it for 24 hours, demold it, and obtain the blood-derived GeLMA-lignin biomimetic hydrogel scaffold.

[0085] The blood-derived GeLMA-lignin biomimetic hydrogel scaffold, through "structural biomimicry" and "functional biomimicry," simulates the structure of cancellous bone while enhancing the mechanical properties of the material, maintaining the osteogenic space, and reducing the level of local reactive oxygen species by relying on the antioxidant properties of lignin as LIG / PRF nanoparticles degrade. Furthermore, with degradation, the growth factors in PRF are released in the optimal physiological ratio, synergistically promoting bone regeneration.

[0086] Please see Figure 2-3 LIG nanoparticles and LIG / PRF nanoparticles form regular spheres with a diameter of approximately 140 nm.

[0087] Please see Figure 4-7 It can be seen that the blood-derived GeLMA-lignin biomimetic hydrogel scaffold exhibits an anisotropic biomimetic structure as a whole;

[0088] Please see Figure 8 It can be seen that the addition of lignin nanoparticles to the simple GeLMA hydrogel scaffold significantly increases its Young's modulus, thus greatly improving its mechanical strength.

[0089] Please see Figure 9 The characteristic absorption peaks of GeLMA can be seen in lignin nanoparticles;

[0090] Please see Figure 10 Different concentrations of LIG / PRF nanoparticles (L: 0.8 mg / ml, M: 1 mg / ml, H: 1.2 mg / ml) were added to the blood-derived GeLMA-lignin biomimetic hydrogel scaffold. As the concentration of nanoparticles reached a certain level, cell activity began to decrease. Therefore, 1 mg of LIG / PRF nanoparticles was added to 1 ml of GeLMA solution for subsequent experiments.

[0091] Please see Figure 11 The results showed that all groups exhibited a proliferative trend, while the proliferation of BMSCs in the GEL / LNP / PRF group was significantly higher than that in other groups (Note: BMSCs are bone marrow mesenchymal stem cells).

[0092] Please see Figure 12 After co-culturing the material extract with BMSCs for 1, 4, and 7 days, the staining was observed using a confocal microscope. The results showed the same proliferation pattern as that observed with CCK-8. Please refer to [link to relevant documentation]. Figure 13 The staining was observed using a confocal microscope. It was found that the GEL / LNP / PRF group showed the best cell proliferation (where Calcein is calcein, green represents live cells, and red represents dead cells).

[0093] Please see Figure 14-15 ,in Figure 14 (LPS in the figure is lipopolysaccharide) is a fluorescent staining of ROS (reactive oxygen species) levels in BMSCs (bone marrow mesenchymal stem cells), intended to indicate the material's ability to scavenge reactive oxygen species; Figure 15 It is aimed at Figure 14 The quantitative analysis of fluorescence intensity (bar chart), the scaffold's ability to scavenge reactive oxygen species, and quantitative analysis show that, compared with the GEL group, the GEL / LNP group and the GEL / LNP / PRF group with added lignin nanoparticles have stronger reactive oxygen species scavenging abilities than the GEL group, indicating that the addition of lignin nanoparticles enhances the overall antioxidant capacity of the scaffold material.

[0094] Please see Figure 16 The results showed that the GEL / LNP / PRF group had more HUVEC cells migrating compared to the GEL group and the GEL / LNP group.

[0095] Please see Figure 17 The results showed that HUVECs in the GEL / LNP / PRF group formed more tubular structures compared with the GEL group and the GEL / LNP group.

[0096] Please see Figure 18 The results showed that HUVEC cells in the GEL / LNP / PRF group migrated significantly faster than those in the GEL group and the GEL / LNP group. After 24 hours, the cell scratches in the GEL / LNP / PRF group were almost completely closed.

[0097] Please see Figures 19-20(In the image, HIF-1α is an abbreviation for hypoxia-inducible factor-1α, a cytokine related to angiogenesis. Green represents the expression of this protein; the brighter the fluorescence, the higher the protein expression level. Dapi, short for 4',6-diamidinyl-2-phenylindole, is a blue fluorescent dye that can penetrate cell membranes and binds highly specifically and tightly to DNA in the cell nucleus (especially regions rich in adenine-thymine). Its core function is to label the cell nucleus, hence Dapi represents nuclear staining and is blue. Phalloidin, whose core function is to label F-actin filaments, represents cytoskeleton staining and is red. Merged is a combined overlay of HIF-1α, Dapi, and Phalloidin.) This is an immunofluorescence image showing the protein expression of angiogenesis-related cytokines within cells after co-culturing cells and materials (brighter fluorescence indicates higher protein expression levels). Figure 19 It is a VEGF cytokine. Figure 20 The images show the immunofluorescence of VEGF and HIF-1α, cytokines associated with angiogenesis, in the GEL, GEL / LNP, and GEL / LNP / PRF groups. The results show that the fluorescence intensity of VEGF and HIF-1α, proteins related to angiogenesis, was significantly increased in the GEL / LNP / PRF group compared with the GEL and GEL / LNP groups.

[0098] Please see Figure 21-22 These are the expression levels of mRNAs of factors related to angiogenesis, which are the results of RT-qPCR. Figure 21 For VEGF, Figure 22 The HIF-1α assay, similarly intended to demonstrate the material's ability to promote angiogenesis, uses a different method (immunofluorescence to demonstrate protein levels, RT-qPCR to demonstrate gene levels). The results of q-PCR experiments on the angiogenesis-related cytokines VEGF and HIF-1α in GEL, GEL / LNP, and GEL / LNP / PRF scaffolds were presented. To further verify the vascularization potential of different scaffolds, we used RT-qPCR to detect the expression of vascularization-related genes at specific time points. The results showed that the GEL / LNP / PRF group had higher VEGF and HIF-1α gene expression levels, which were significantly different from the blank group, GEL group, and GEL / LNP group.

[0099] Please see Figure 23 After co-culturing different groups of scaffolds with rBMSCs for 7 days, alkaline phosphatase staining showed that the staining density of the GEL / LNP / PRF group was higher than that of the blank group, GEL group, and GEL / LNP group.

[0100] Please see Figure 24 After co-culturing different groups of scaffolds with rBMSCs for 21 days, the results of alizarin red staining showed that the calcium nodule density of the GEL / LNP / PRF group was significantly increased compared with the GEL group, GEL / LNP group and blank group.

[0101] Please see Figure 25-26 Immunofluorescence images of protein expression of classic osteogenic cytokines within cells after co-culture of cells and materials (brighter fluorescence indicates higher protein expression levels), among which... Figure 25 It is RUNX-2 cytokine. Figure 26 The OCN cytokine assay was designed to demonstrate that the developed material promotes bone formation. Immunofluorescence of the osteogenic-related cytokine OCN RUNX-2 in GEL, GEL / LNP, and GEL / LNP / PRF scaffolds was used to assess the expression levels of the corresponding osteogenic proteins. Runx2 and OCN-related proteins showed stronger fluorescence intensity in rBMSCs co-cultured with the GEL / LNP / PRF scaffold (Runx-2 is an abbreviation for Runt-related transcription factor 2; green in the figure represents this protein expression, with stronger fluorescence indicating higher protein expression). Dapi, short for 4',6-diamidinyl-2-phenylindole, is a blue fluorescent dye that can penetrate cell membranes and is used to label the cell nucleus; Dapi represents nuclear staining and is blue. Phalloidin is a phalloidin peptide used to label F-actin filaments and represents cytoskeleton staining, and is red. Figure 25 Merged is the merged and overlapping graph of Runx-2, Dapi, and Phalloidin. Figure 26 (Merged is the merged overlapping graph of OCN, Dapi, and Phalloidin).

[0102] Please see Figure 27-28 The expression levels of mRNAs of classic cytokines related to bone formation, which are the results of RT-qPCR. Figure 27 For RUNX-2, Figure 28 For OCN, the same purpose is to demonstrate that the material has the ability to promote angiogenesis, but with different methods of demonstration (immunofluorescence to demonstrate protein levels, RT-qPCR to demonstrate gene levels). The RT-qPCR results of osteogenic-related cytokines OCN RUNX-2 in GEL, GEL / LNP, and GEL / LNP / PRF scaffolds showed that the gene expression levels of osteogenic-related proteins RUNX-2 and OCN in BMSCs on the surface of GEL / LNP / PRF scaffolds were significantly higher than those in other groups, proving that GEL / LNP / PRF scaffolds can effectively promote osteogenic differentiation.

[0103] In the experiments of this invention, comparative analysis was conducted at the protein level. The data shown in the figures were analyzed using statistical software, and the results for each group are expressed as "mean ± standard deviation". At the same time point, the relative expression levels of OCN mRNA between different treatment groups were compared using one-way ANOVA, with a significance level set at α = 0.05. All tests were two-tailed. When homogeneity of variance was established, pairwise comparisons were further performed using the LSD post-hoc test. The asterisks in the figures represent different levels of statistical significance: one asterisk indicates P < 0.05, indicating a statistically significant difference; two asterisks indicate P < 0.01, indicating a significant difference; three asterisks indicate P < 0.001, indicating an extremely significant difference. If P ≥ 0.05, no asterisk is used, indicating no statistically significant difference.

[0104] Although the present invention has been described above with reference to embodiments, various modifications can be made and components can be replaced with equivalents without departing from the scope of the invention. In particular, as long as there is no structural conflict, the features in the disclosed embodiments can be combined with each other in any manner. The lack of an exhaustive description of these combinations in this specification is merely for the sake of brevity and resource conservation. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A method for preparing a blood-derived GeLMA-lignin biomimetic hydrogel scaffold, characterized by, The method comprises the following steps: S1: preparing GeLMA hydrogel precursor: adding 5 g pigskin gelatin in PBS, heating in water bath, stirring to dissolve, adding methacrylic anhydride, stirring, preparing GeLMA hydrogel precursor solution, adjusting PH=7, dialysis, freeze-drying; S2: preparing lignin nanoparticles: adding alkaline lignin in methanol, stirring to fully dissolve, centrifuging, removing insoluble impurities at the bottom, adding deionized water, stirring, centrifuging to collect precipitate, freeze-drying; S3: preparing PRF powder: taking SD rat arterial blood to prepare PRF powder; S4: preparing LIG / PRF nanoparticles; S5: preparing blood-derived GeLMA-lignin biomimetic hydrogel scaffold: S5.1: weighing 120 mg of GeLMA hydrogel precursor and 1 mg of LIG / PRF nanoparticles, dissolving in 1 ml of PBS solution containing 100 μl of LAP, avoiding light, 37℃ water bath, and oscillating until fully dissolved; S5.2: transferring the solution into a red copper mold, precooling the ice mold machine to 45℃, placing the mold on the cold source of the ice mold machine, irradiating with ultraviolet light while the ice crystals crystallize, and light curing after the solution is completely frozen, continuing to freeze in the ice mold for 2 hours; S5.3: taking the sample, freezing in liquid nitrogen for 10 seconds, then-80℃ overnight, freeze-drying for 24 hours, demolding, and preparing blood-derived GeLMA-lignin biomimetic hydrogel scaffold.

2. A process for the preparation of a blood derived GeLMA-lignin biomimetic hydrogel scaffold as claimed in claim 1, wherein, The adding method of methacrylic anhydride in S1 is: slowly and dropwise adding at a rate of 0.3 ml / min, and the total amount is 6 ml.

3. A process for the preparation of a blood derived GeLMA-lignin biomimetic hydrogel scaffold according to claim 1, characterized in that, The dialysis method in S1 is: transferring the GeLMA hydrogel precursor solution after adjusting PH into a dialysis bag with a molecular weight cut-off of 8000 D-14000 D, dialyzing in deionized water at 40℃ for seven days, and replacing the deionized water every three hours during this period.

4. The method of claim 1, wherein the blood-derived GeLMA-lignin biomimetic hydrogel scaffold is prepared by the steps of: The specific method of S3 is: S3.1: taking 5 ml of SD rat arterial blood in an anticoagulant-free centrifuge tube, centrifuging at 3000 rpm for 12 minutes; S3.2: after centrifuging, layering, taking the upper part, placing between two gauze, pressing to remove water, and preparing PRF film; S3.3: after freeze-drying for 24 hours, fully grinding the PRF film to prepare PRF powder.

5. A process for the preparation of a blood derived GeLMA-lignin biomimetic hydrogel scaffold according to claim 1, characterized in that, The specific method of S4 is: S4.1: resuspending 10 mg of lignin nanoparticles in 100 μl of PBS; S4.2: then adding 3 mg of PRF powder to the resuspended lignin nanoparticle solution, fully dissolving, and incubating at 37℃ for 24 hours; S4.3: placing the sample at-80℃ overnight, then freeze-drying for 24 hours to obtain LIG / PRF nanoparticles.

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