A preparation method of a blood-derived gelma-lignin biomimetic hydrogel scaffold

Abstract

The application 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 biomimetic hydrogel scaffold. The method comprises the following steps: S1: preparing a GeLMA hydrogel precursor; S2: preparing lignin nanoparticles; S3: preparing PRF powder; S4: preparing LIG / PRF nanoparticles; and S5: preparing the blood-derived GeLMA-lignin biomimetic hydrogel scaffold. The single cold source ice mold technology of the application can control the pore direction, and the structure is similar to cancellous bone in height. The lignin nanoparticles realize the triple functions of mechanical enhancement, antioxidant protection and growth factor release. The PRF is derived from autologous blood, has the best physiological proportion and synergistic effect, and avoids the proportion imbalance and immune rejection possibly caused by the addition of exogenous factors. The single cold source process has strong controllability and has industrialization potential.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical materials and bone tissue engineering, and particularly relates to a preparation method of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold. BACKGROUND

[0002] In clinical bone defect repair, autologous bone transplantation is still widely used, but this method has many limitations: (1) a second bone harvesting operation area needs to be created, increasing the trauma and surgical risk of the patient; (2) the amount of bone harvested is limited, and a second operation is often required to remove the fixation device; (3) allogeneic or artificial bone transplantation has problems of immune rejection and low osteogenic efficiency. Therefore, it is of great clinical significance to develop a new type of bone tissue engineering scaffold with good biocompatibility, degradable absorption, and the ability to promote osteogenesis.

[0003] In recent years, GeLMA (hydrogel material) has been widely used in the construction of tissue engineering scaffolds due to its good biocompatibility and cell adhesion. However, the main defects of GeLMA hydrogel are insufficient mechanical strength, poor antioxidant performance, and limited biological activity, which limit its application in weight-bearing bone repair. At the same time, in clinical practice, the increase in the level of reactive oxygen species (ROS) during the healing process of the defect after surgery can cause oxidative stress and cell damage. Oxidative stress can inhibit mesenchymal cell osteogenic differentiation by inhibiting the Hedgehog signaling pathway, and can transfer beta-Catenin to FoxO-mediated transcription, thereby antagonizing the Wnt signaling pathway in osteoblast precursors, which has a great impact on the final osteogenesis effect. Therefore, how to improve the mechanical properties of GeLMA and endow it with antioxidant and vascularization-promoting ability, and synergistically promote osteogenesis, is the key difficulty in current research.

[0004] To solve these problems, we provide a preparation method of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold. SUMMARY

[0005] This section aims to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of the specification to avoid obscuring the purpose of this section, abstract and title, and such simplifications or omissions cannot be used to limit the scope of the present application.

[0006] To solve the above technical problems, according to one aspect of the present application, the present application provides the following technical scheme:

[0007] A preparation method of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold, comprising the following steps:

[0008] S1: Preparation of GeLMA hydrogel precursor: add 5g pigskin gelatin in PBS, heat in water bath, stir to dissolve, add methacrylic anhydride, stir, prepare GeLMA hydrogel precursor solution, adjust PH=7, dialysis, freeze-drying;

[0009] S2: Preparation of lignin nanoparticles: add alkaline lignin into methanol, stir to fully dissolve, centrifuge, remove insoluble impurities at the bottom, add deionized water, stir, centrifuge to collect precipitate, freeze-drying;

[0010] S3: Preparation of PRF powder: take SD rat arterial blood to prepare PRF powder;

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

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

[0013] S5.1: weigh 120mg of GeLMA hydrogel precursor and 1mg of LIG / PRF nanoparticles, dissolve in 1ml of PBS solution containing 100ul of LAP, avoid light, 37℃ water bath, shake until fully dissolved;

[0014] S5.2: transfer the solution into red copper molds respectively, pre-cool the ice mold machine to 45℃, then place the molds on the cold source of the ice mold machine, irradiate with ultraviolet light while the ice crystals crystallize, completely freeze the solution, continue to freeze in the ice mold machine for 2 hours;

[0015] S5.3: take the sample, freeze in liquid nitrogen for 10 seconds, then-80℃ overnight, freeze-drying for 24 hours, demold, prepare blood-derived GeLMA-lignin biomimetic hydrogel scaffold.

[0016] As a preferred scheme of the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold, in S1, the methacrylic anhydride is added at a rate of 0.3ml / min, slowly and drop by drop, and the total amount is 6ml.

[0017] As a preferred scheme of the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold, in S1, the dialysis method is as follows: transfer the GeLMA hydrogel precursor solution with adjusted PH into a dialysis bag with a molecular weight cut-off of 8000D-14000D, dialyze in deionized water at 40℃ for seven days, and replace the deionized water every three hours during this period.

[0018] As a preferred scheme of the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold, in the specific method of S3, the specific method is S3.1: 5ml of SD rat arterial blood is taken in a centrifuge tube without anticoagulant, and centrifuged at 3000rpm for 12 minutes;

[0019] S3.2: After centrifugation, separate the layers, take the upper part, and place it between two gauze, and press to remove water to obtain a PRF film;

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

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

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

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

[0024] Compared with the prior art, the present application has the beneficial effects that: 1. stronger structural biomimicry: single cold source ice template technology can control the pore direction, and the structure is highly similar to cancellous bone; 2. multifunctional integrated design: mechanical reinforcement, antioxidant protection and growth factor release are realized by lignin nanoparticles; 3. endogenous growth factor advantage: PRF is derived from autologous blood, has the best physiological ratio and synergistic effect, and avoids the imbalance of proportion and immune rejection caused by the addition of exogenous factors; 4. degradable and no secondary surgery: the scaffold gradually degrades and absorbs in the body, without the need for secondary removal, reducing patient pain; 5. simple preparation and scalability: the single cold source process has strong controllability and has industrialization potential. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the present application will be described in detail below in combination with the drawings and detailed embodiments. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor. Among them:

[0026] Figure 1 Flow chart of the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present application;

[0027] Figure 2 LIG NP TEM image for the preparation method of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present application;

[0028] Figure 3 LIG / PRF TEM image for the preparation method of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present application;

[0029] Figure 4 Synthetic GeLMA-lignin biomimetic hydrogel scaffold image for the preparation method of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present application;

[0030] Figure 5 Microstructure image of the scaffold for the preparation method of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present application;

[0031] Figure 6 Scaffold cross-section SEM 10 times picture for the preparation method of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present application;

[0032] Figure 7 Scaffold cross-section SEM 200 times picture for the preparation method of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present application;

[0033] Figure 8 Young's modulus of GeLMA / LNP / PRF containing 0.8 mg / ml LIG / PRF NP, 1 mg / ml LIG / PRF NP, 1.2 mg / ml LIG / PRF NP concentration for the preparation method of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present application (L: 0.8 mg / ml LIG / PRF nanoparticles are added to the scaffold; M: 1 mg / ml LIG / PRF nanoparticles are added to the scaffold; H: 1.2 mg / ml LIG / PRF nanoparticles are added to the scaffold);

[0034] Figure 9 Fourier infrared spectrum of GeLMA GeLMA / LNP GeLMA / LNP / PRF scaffold for the preparation method of a blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present application;

[0035] Figure 10A CCK-8 biocompatibility detection diagram of GeLMA / LNP / PRF with 0.8 mg / ml LIG / PRF NP, 1 mg / ml LIG / PRF NP and 1.2 mg / ml LIG / PRF NP concentrations for the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the application (L: the LIG / PRF nanoparticles of 0.8 mg / ml are added to the scaffold; M: the LIG / PRF nanoparticles of 1 mg / ml are added to the scaffold; H: the LIG / PRF nanoparticles of 1.2 mg / ml are added to the scaffold);

[0036] Figure 11 A metabolic activity diagram of CON (blank group), GeL (pure GeL scaffold), GEL / LNP (scaffold with lignin nanoparticles added to GeLMA) and GEL / LNP / PRF (scaffold with PRF-loaded growth factor-containing lignin nanoparticles added to GeLMA hydrogel) scaffolds on BMSCs detected by the CCK-8 method for the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the application;

[0037] Figure 12 A staining diagram observed by a confocal microscope after the material extract and BMSCs are co-cultured for 1 day, 4 days and 7 days for the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the application;

[0038] Figure 13 A staining diagram after live and dead staining of bone marrow mesenchymal stem cells seeded on the scaffold for 1 day, 4 days and 7 days under a confocal microscope for the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the application;

[0039] Figure 14 A fluorescence staining diagram of the in-vivo ROS (reactive oxygen species) level of bmsc (bone marrow mesenchymal stem cells) for the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the application;

[0040] Figure 15 A diagram for the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the application; Figure 14 A quantitative analysis column chart of fluorescence intensity;

[0041] Figure 16 A huvecs Transwell experiment result diagram of GEL, GEL / LNP and GEL / LNP / PRF scaffolds for the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the application;

[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 26An immunofluorescence image of protein expression of OCN cell factor related to bone formation in the cell after cell and material co-culture according to the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present application;

[0052] Figure 27 The mRNA expression amount of RUNX-2 cell factor related to bone formation according to the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present application;

[0053] Figure 28 The mRNA expression amount of OCN cell factor related to bone formation according to the preparation method of the blood-derived GeLMA-lignin biomimetic hydrogel scaffold of the present application. DETAILED DESCRIPTION

[0054] In order to make the above objectives, features and advantages of the present application more apparent and comprehensible, the specific embodiments of the present application will be described in detail below with reference to the accompanying drawings.

[0055] Secondly, the present application is described in detail in combination with the schematic diagram, and in the detailed description of the embodiments of the present application, the cross-sectional view of the device structure will be partially enlarged without the general proportion for the convenience of description, and the schematic diagram is only an example, which should not limit the scope of protection of the present application herein. In addition, the three-dimensional spatial dimensions of length, width and depth should be included in the actual manufacture.

[0056] In order to make the objectives, technical solutions and advantages of the present application more apparent and comprehensible, the embodiments of the present application will be described in further detail below with reference to the accompanying drawings.

[0057] The present application introduces lignin nanoparticles loaded with growth factors into the GeLMA hydrogel system, combines single cold source ice template technology, and constructs a bone tissue engineering scaffold with anisotropy, porous biomimetic structure (similar to cancellous bone structure), realizes the unity of "structure biomimicry" and "function biomimicry". The endogenous growth factor group derived from platelet-rich plasma (PRF) is introduced into the scaffold, which is derived from the patient's own blood, has the best physiological ratio and natural synergistic effect, can significantly promote the adhesion, proliferation and differentiation of osteoblasts, and at the same time avoids the immune rejection and proportion imbalance problems that may be caused by heterologous growth factors. Lignin nanoparticles further endow the material with excellent mechanical properties and antioxidant function, so that the scaffold has three functions of mechanical strengthening, antioxidant and biological activity regulation.

[0058] This scaffold has multiple mechanisms: 1. Mechanical reinforcement mechanism: hydrogen bonds or covalent cross-linking between lignin nanoparticles and GeLMA molecular chains increase cross-linking density and elastic modulus, significantly improving the scaffold's compression resistance 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 release mechanism: PRP loaded in the scaffold is derived from autologous blood, containing various growth factors such as PDGF, TGF-β, VEGF, and IGF, which coexist in the best physiological ratio and have a natural synergistic effect.

[0059] Lignin nanoparticles can act as a sustained-release carrier, allowing controlled release of these endogenous growth factors and avoiding the burst release that occurs when PRF is directly incorporated.

[0060] In this way, growth factors can be released long-term, stably, and in a physiological rhythm within the material, 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: Prepare GeLMA hydrogel precursor:

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

[0064] S1.2: Slowly and dropwise add 6 ml of methacrylic anhydride to the gelatin solution at a rate of 0.3 ml / min, then continue stirring at 500 rpm for 3 hours, and the substitution reaction is complete. The GeLMA hydrogel precursor solution is prepared;

[0065] S1.3: Add a small amount of NaOH solution to the GeLMA hydrogel precursor solution multiple times, and measure the solution pH. When the pH is adjusted to 7, the reaction is terminated;

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

[0067] S1.5: After dialysis, transfer the product to a container, freeze overnight at -80°C, and then freeze-dry for 48 hours. The freeze-dried GeLMA hydrogel precursor is a porous foam-like material.

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

[0069] S2.1: 60 mg of alkaline lignin was added into 10 ml of methanol and magnetically stirred for 2 hours for complete dissolution;

[0070] S2.2: The solution was transferred to a centrifuge tube and after short centrifugation at 800 rpm for 2 minutes, the bottom insoluble impurities were removed;

[0071] S2.3: The solution was transferred to a beaker and while magnetically stirring, deionized water was slowly added to the solution, 6 ml of deionized water for every 2 ml of lignin solution, after all the water was added, stirring was continued for 10 minutes;

[0072] S2.4: After completion of stirring, the sample was centrifuged at 10000 x g for 10 minutes, the precipitate was collected, stored at -80°C overnight, and then freeze-dried for 24 hours to obtain lignin nanoparticles;

[0073] S3: Preparation of PRF powder:

[0074] S3.1: 5 ml of arterial blood of SD rats was taken in a centrifuge tube without anticoagulant, and centrifuged at 3000 rpm for 12 minutes;

[0075] S3.2: After centrifugation, the upper layer was taken and placed between two gauze, and pressed to remove water to obtain PRF membrane;

[0076] S3.3: The PRF membrane was freeze-dried for 24 hours, and then ground to obtain PRF powder;

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

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

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

[0080] S4.3: The sample was stored at -80°C 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: 120 mg of GeLMA hydrogel precursor and 1 mg of LIG / PRF nanoparticles were weighed and dissolved in 1 ml of PBS solution containing 100 μl of LAP, protected from light, and shaken at 37°C water bath until complete dissolution;

[0083] S5.2: Transfer the solution into the red copper mold respectively, after the ice mold machine is pre-cooled to 45℃, place the mold on the cold source of the ice mold machine, irradiate with ultraviolet light (405nm) while the ice crystals crystallize, and freeze the solution completely. Continue to freeze in the ice mold for 2 hours after the solution is completely frozen;

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

[0085] Through the blood-derived GeLMA-lignin biomimetic hydrogel scaffold, the structure of cancellous bone is simulated through "structure simulation" and "function simulation", the mechanical properties of the material are enhanced, the osteogenesis space is maintained, and with the degradation of LIG / PRF nanoparticles, the local reactive oxygen level is reduced due to the antioxidant property of lignin, and with the degradation, the growth factors in PRF are released at the optimal physiological ratio, which synergistically promotes bone regeneration.

[0086] Please refer to Figures 2-3 , the LIG nanoparticles and the LIG / PRF nanoparticles form regular spherical shapes with a diameter of about 140nm;

[0087] Please refer to Figures 4-7 , it can be seen that the blood-derived GeLMA-lignin biomimetic hydrogel scaffold presents an anisotropic biomimetic structure as a whole;

[0088] Please refer to Figure 8 , it can be seen that the Young's modulus of the pure GeLMA hydrogel scaffold is significantly increased after the addition of lignin nanoparticles, which fully improves the mechanical strength;

[0089] Please refer to Figure 9 , it can be seen that the lignin nanoparticles and the GeLMA characteristic absorption peaks;

[0090] Please refer to Figure 10 , the blood-derived GeLMA-lignin biomimetic hydrogel scaffold is added with different concentrations of LIG / PRF nanoparticles (L: 0.8mg / ml, M: 1mg / ml, H: 1.2mg / ml), and with the addition of nanoparticles to a certain extent, the cell activity begins to decrease, so 1mg of LIG / PRF nanoparticles is added to 1ml of GeLMA solution for subsequent experiments;

[0091] Please refer to Figure 11 , the results show that all groups show a proliferation trend, and the proliferation of BMSCs in the GEL / LNP / PRF group is significantly higher than that in the other groups (note: BMSCs are bone marrow mesenchymal stem cells);

[0092] Please refer toFigure 12 After the material leaching solution was co-cultured with BMSCs for 1 day, 4 days and 7 days, the staining was observed by confocal microscope, and the results showed that the same proliferation as the CCK-8 results could be observed; see Figure 13 The staining was observed by confocal microscope, and it could be seen that the GEL / LNP / PRF group presented the best cell proliferation (wherein, Calcein is calcein, the imaging of green cells in the figure is green, and PI is the imaging of dead cells, which is red;

[0093] See Figures 14-15 Among them Figure 14 The fluorescence staining of bmsc (bone marrow mesenchymal stem cells) in vivo ros (reactive oxygen species) level, which is intended to show the ability of the material to remove reactive oxygen species; Figure 15 is for Figure 14 The fluorescence intensity quantitative analysis column chart, the ability of the scaffold to remove reactive oxygen species and quantitative analysis, it can be seen that compared with the GEL group, the reactive oxygen species removal ability of the GEL / LNP group and the GEL / LNP / PRF group added with lignin nanoparticles is stronger than that of the GEL group, which shows that the addition of lignin nanoparticles to the scaffold material enhances the overall antioxidant capacity of the scaffold material;

[0094] See Figure 16 The results show that compared with the GEL group and the GEL / LNP group, the HUVEC cell migration amount of the GEL / LNP / PRF group is more;

[0095] See Figure 17 The results show that compared with the GEL group and the GEL / LNP group, the GEL / LNP / PRF group forms more tubular structures;

[0096] See Figure 18 The results show that compared with the GEL group and the GEL / LNP group, the HUVEC cell migration speed of the GEL / LNP / PRF group is significantly faster, and the cell scratch of the GEL / LNP / PRF group is almost completely closed after 24 hours;

[0097] See Figures 19-20(HIF-1a is the abbreviation of hypoxia-inducible factor-1a, which is a cytokine related to angiogenesis, and green represents the expression of this protein, and the brighter the brightness, the more protein expression; Dapi is 4', 6-diamidino-2-phenylindole, which is a blue fluorescent dye that can penetrate the cell membrane, can highly specifically bind to DNA in the cell nucleus (especially the adenine-thymine-rich region), and the core function is to label the nucleus, so Dapi represents the nucleus staining, which is blue; Phalloidin is a phalloidin, and the core function is to label F-actin filaments, which represents the cytoskeleton staining, which is red; Merged is the merged image of HIF-1a, Dapi, and Phalloidin), after co-culturing cells with materials, immunofluorescence image of protein expression of cytokines related to angiogenesis in cells (the brighter the fluorescence, the more protein expression), among which Figure 19 VEGF cytokine, Figure 20 HIF-1a cytokine, which is intended to prove that the developed material has the ability to promote angiogenesis, and the immunofluorescence images of VEGF and HIF-1a cytokines related to angiogenesis in the GEL group, the GEL / LNP group and the GEL / LNP / PRF group, the results show that the fluorescence intensity of VEGF and HIF-1a proteins related to angiogenesis in the GEL / LNP / PRF group is significantly higher than that in the GEL group and the GEL / LNP group;

[0098] Please refer to Figures 21-22 , which is the mRNA expression of factors related to angiogenesis, that is, the results of RT-qPCR, Figure 21 VEGF, Figure 22 HIF-1a, which is also intended to prove that the material has the ability to promote angiogenesis, but the proving means is different (immunofluorescence proves the protein level, and RT-qPCR proves the gene level), and the q-PCR experimental results of VEGF and HIF-1a cytokines related to angiogenesis in the GEL, GEL / LNP and GEL / LNP / PRF scaffolds, in order to further verify the vascularization potential of different scaffolds, we detected the expression of vascularization-related genes at a certain time point by RT-qPCR. The results show that the expression of VEGF and HIF-1a genes in the GEL / LNP / PRF group is higher, which has a significant difference with the blank group, the GEL group and the GEL / LNP group;

[0099] Please refer to Figure 23 After co-culturing different groups of scaffolds with rBMSCs for 7 days, alkaline phosphatase staining shows that the staining density of the GEL / LNP / PRF group is higher than that of the blank group, the GEL group and the GEL / LNP group;

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

[0101] Please refer to Figures 25-26 After co-culturing cells with materials, the immunofluorescence images of protein expression of the classic cell factors related to osteogenesis in cells (the brighter the fluorescence, the more protein expression), in which Figure 25 is the RUNX-2 cell factor, Figure 26 is the OCN cell factor, are intended to prove that the developed material has the ability to promote bone formation, the immunofluorescence of the cell factors OCN and RUNX-2 related to osteogenesis of the GEL, GEL / LNP and GEL / LNP / PRF scaffolds, the expression level of the corresponding protein of osteogenesis was evaluated by immunofluorescence staining, and the fluorescence intensity of the Runx2 and OCN related proteins in the rBMSCs co-cultured with the GEL / LNP / PRF scaffold was stronger (in which, Runx-2 is the abbreviation of Runt-related transcription factor 2, green in the figure represents the expression of this protein, and the brighter the brightness, the more protein expression; Dapi is a blue fluorescent dye that can penetrate the cell membrane, used to label the cell nucleus, Dapi represents cell nucleus staining, which is blue; Phalloidin is a phalloidin used to label F-actin filaments, representing cytoskeleton staining, which is red; Figure 25 Merged in which Runx-2, Dapi, Phalloidin are merged and overlapped; Figure 26 Merged in which OCN, Dapi, Phalloidin are merged and overlapped);

[0102] Please refer to Figures 27-28 The expression level of the mRNA of the classic cell factors related to bone formation, that is, the results of RT-qPCR, Figure 27 is RUNX-2, Figure 28 is OCN, which is also intended to prove that the material has the ability to promote blood vessel formation, but the proving means is different (immunofluorescence proves the protein level, and RT-qPCR proves the gene level), the RT-qPCR results of the cell factors OCN and RUNX-2 related to osteogenesis of the GEL, GEL / LNP and GEL / LNP / PRF scaffolds, the results show that the gene expression level of the osteogenesis related proteins RUNX-2 and OCN of the BMSCs on the surface of the GEL / LNP / PRF scaffold is obviously higher than that of the other groups, proving that the GEL / LNP / PRF scaffold can effectively promote osteogenic differentiation.

[0103] In the experiment of the present application, comparative analysis is carried out at the protein level, and the data shown in the drawings are analyzed by statistical software, and the results of each group are represented by "mean ± standard deviation". At the same time point, the relative expression of OCN mRNA in different treatment groups is compared by one-way ANOVA, and the test level is set as α = 0.05, and all are two-sided tests; when the homogeneity of variance is established, further LSD post-hoc test is used for pairwise comparison, and the asterisks in the figure represent different statistical significance levels: one star indicates P < 0.05, and the difference is statistically significant; two stars indicate P < 0.01, and the difference is significant; three stars indicate P < 0.001, and the difference is extremely significant, and if P ≥ 0.05, no star is marked, indicating that the difference is not statistically significant.

[0104] Although the present application has been described above with reference to the embodiments, various modifications can be made to it and components thereof can be replaced with equivalents without departing from the scope of the present application. In particular, each feature in the embodiments disclosed in the present specification can be used in combination with any of the other features unless there is a structural conflict. The combinations of these features are not exhaustively described in the present specification only for the purpose of omitting the description and saving resources. Therefore, the present application 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 precipitates, freeze-drying; S3: preparing PRF powder: taking arterial blood of an SD rat to prepare PRF powder: S3.1: taking 5 ml of arterial blood of an SD rat in a centrifuge tube without anticoagulant, centrifuging at 3000 rpm for 12 minutes; S3.2: after centrifuging, separating layers, taking the upper part, placing between two gauzes, pressing to remove water, preparing PRF film; S3.3: after freeze-drying the PRF film for 24 hours, fully grinding to prepare PRF powder; S4: preparing LIG / PRF nanoparticles: S4.1: resuspending 10 mg of lignin nanoparticles in 100 μl of PBS; S4.2: then adding 3 mg of PRF powder into 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; S5: preparing blood-derived GeLMA-lignin biomimetic hydrogel scaffold: S5.1: taking 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, oscillating until fully dissolved in a 37℃ water bath; S5.2: transferring the solution into a red copper mold, fully precooling the ice mold machine to 45℃, then placing the mold on the cold source of the ice mold machine, irradiating with ultraviolet light while the ice crystals are crystallizing, completely freezing the solution, continuing to freeze in the ice mold for 2 hours; S5.3: taking the sample, freezing in liquid nitrogen for 10 seconds, then placing at -80℃ overnight, freeze-drying for 24 hours, demolding, to prepare a 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 the 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 as claimed in claim 1, wherein, 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 changing the deionized water every three hours during this period.

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