A three-dimensional cell scaffold based on nandina domestica leaf vein and a preparation method and application thereof

Abstract

The application discloses a three-dimensional cell scaffold based on a leaf vein of nandina domestica and a preparation method and application thereof, relates to the technical field of biomedical materials and tissue engineering, and discloses the following steps: taking the leaf vein of nandina domestica as a base material, and preparing the scaffold through alkali boiling, sodium periodate selective oxidation and amino-silica / chitosan composite modification. The scaffold retains the three-dimensional structure of the natural leaf vein completely, and significantly improves the cell compatibility of the material. Cell experiments prove that the scaffold has no cytotoxicity, can support the three-dimensional growth of mesenchymal stem cells and PC12 cells and the like with high survival rate, and effectively promotes cell proliferation. The method is simple, green, environmentally-friendly and low in cost, and the obtained scaffold has wide application prospects in the fields of tissue engineering, regenerative medicine and drug screening and the like.

Description

Technical Field

[0001] This invention relates to the fields of biomedical materials and tissue engineering technology, and more specifically to a three-dimensional cell scaffold based on the veins of Nandina domestica leaves, its preparation method, and its application. Background Technology

[0002] Three-dimensional cell culture technology plays an increasingly important role in tissue engineering, regenerative medicine, drug screening, and tumor research because it can better simulate the cell growth microenvironment in vivo. An ideal three-dimensional scaffold should have good biocompatibility, suitable pore structure, surface properties conducive to cell adhesion and proliferation, and be easy to prepare and functionalize.

[0003] Currently, commonly used three-dimensional scaffolds mainly include natural polymer materials (such as collagen, gelatin, chitosan, and sodium alginate), synthetic polymer materials (such as PLA and PLGA), and their composites. Natural materials have good biocompatibility, but their mechanical properties and structural stability are often insufficient; synthetic materials, although having controllable mechanical properties, often suffer from poor hydrophilicity and degradation products that may cause inflammatory reactions. In recent years, decellularized tissue matrices and biomimetic mineralization materials have also been extensively studied, but they still face challenges such as limited sources, high costs, or complex preparation processes.

[0004] Plant-derived materials, especially their cellulose skeletons, possess advantages such as wide availability, low cost, renewability, biodegradability, and natural porosity, making them a potential substrate for three-dimensional scaffolds. Plant vein networks, as a natural cellulose-based three-dimensional porous structure, can theoretically provide growth space for cells. However, untreated plant vein surfaces are chemically inert, lack cell recognition sites, and different plant varieties exhibit significant differences in pore size, pore connectivity, and structural stability, meaning not all are suitable for cell culture. Therefore, screening specific plant veins and effectively modifying their surface through biofunctionalization are crucial for their successful application in three-dimensional cell culture.

[0005] In existing technologies, the modification of plant scaffolds is mostly concentrated on single chemical treatments or simple coatings, such as using only alkali treatment to activate the surface, or coating a single protein through physical adsorption. The stability and comprehensiveness of the modified properties need to be improved.

[0006] Therefore, how to organically combine the ideal structure of natural leaf veins with artificially modified biological functions through a simple and feasible process to prepare a three-dimensional cell scaffold with stable performance and wide applicability is a technical problem that needs to be solved in this field. Summary of the Invention

[0007] In view of this, the present invention provides a three-dimensional cell scaffold based on Nandina domestica leaf veins, its preparation method, and its application, overcoming the shortcomings of the prior art. It provides a three-dimensional cell scaffold based on Nandina domestica leaf veins with excellent biocompatibility, effectively supporting three-dimensional cell growth and proliferation, and also provides its preparation method and application. Through specific screening and a series of modifications, the present invention achieves an effective combination of the structural advantages of natural leaf veins and surface functionalization.

[0008] To achieve the above objectives, this application adopts the following technical solution:

[0009] The primary objective of this application is to provide a method for preparing a three-dimensional cell scaffold based on the leaf veins of Nandina domestica, comprising the following steps:

[0010] S1: Pretreatment: Select Nandina domestica leaf veins and immerse them in a 5% sodium hydroxide solution. Boil at 100℃ for 5-7 minutes for pretreatment to remove some lignin, hemicellulose, pectin, and surface wax impurities from the leaf veins, and to activate the hydroxyl groups on the cellulose surface. After treatment, remove the leaf veins, wash them repeatedly with deionized water until neutral, and then dry them for later use.

[0011] S2: Selective oxidation: The pretreated Nandina domestica leaf veins are immersed in a sodium periodate solution for selective oxidation. Sodium periodate oxidation selectively breaks the carbon-carbon bonds between C2 and C3 on the cellulose glucose units, converting them into two active aldehyde groups. After the oxidation reaction is complete, the leaf veins are removed, thoroughly washed with deionized water to remove residual sodium periodate, and then dried.

[0012] S3: Composite Modification: Aminated silica was mixed with a 1.5% (w / w) chitosan solution, resulting in a 1:2 (w / w) mass ratio of solid aminated silica to solid chitosan. The resulting mixture was then immersed in the mixture and reacted at 50°C for 1.5 hours. During this process, the amino groups on the aminated silica formed Schiff base bonds with the aldehyde groups on the leaf vein cellulose, achieving covalent anchoring. Simultaneously, the chitosan molecular chains, through physical entanglement, hydrogen bonding, and interaction with silica, formed a stable composite coating on the leaf vein surface. After the reaction, the leaf veins were removed, washed to remove unbound components, and freeze-dried to obtain a three-dimensional cellular scaffold for the composite leaf vein.

[0013] As a preferred technical solution, the Nandina domestica leaf veins described in step S1, after pretreatment, exhibit a porous network with a pore size of 50–200 μm and a uniform structure.

[0014] As a preferred technical solution, the mass fraction of the sodium periodate solution in step S2 is 0.01% to 1%; the selective oxidation conditions are as follows: reaction for 6 to 12 hours at room temperature in the dark.

[0015] As a preferred technical solution, the chitosan solution in step S3 is prepared using a 1% (v / v) aqueous solution of glacial acetic acid as a solvent; the aminated silica is added in sol form, and its solid content is 1% to 3%.

[0016] As a preferred technical solution, the mass fraction of the sodium periodate solution in step S2 is 0.1% to 0.5%.

[0017] Another object of this application is to provide a three-dimensional cell scaffold based on the veins of Nandina domestica leaves, prepared by the method described above.

[0018] Another object of this application is to provide the application of the aforementioned three-dimensional cell scaffold in three-dimensional cell culture.

[0019] As a preferred technical solution, the cells include mesenchymal stem cells or PC12 cells.

[0020] As a preferred technical solution, the scaffold has good biocompatibility as demonstrated by CCK-8 assay for cell viability and live / dead cell double staining experiment, and can promote cell proliferation as demonstrated by EdU cell proliferation experiment.

[0021] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) Scientific substrate selection and outstanding structural advantages: This invention, for the first time, systematically compared and screened leaf veins of various common plants such as privet, osmanthus, photinia, and nandina. It creatively discovered and proved that after mild alkali treatment, the natural vascular bundle network of nandina leaf veins can form an ideal three-dimensional structure with a pore size of 50-200 μm, uniform distribution, and excellent connectivity, perfectly simulating the physical topology of the extracellular matrix. This structure does not require complex pore-forming processes and directly provides cells with ready-made growth space and nutrient channels that meet physiological needs. This is the material basis and structural prerequisite for the success of this invention.

[0023] (2) High efficiency and synergistic effect of modification process, stable functional coating: This invention designs a three-step progressive process of "alkali boiling pretreatment - sodium periodate selective oxidation - aminated silica / chitosan composite modification". Alkali boiling can efficiently remove impurities and waxes, and significantly increase the specific surface area and reactive sites of leaf veins. Sodium periodate oxidation accurately introduces active aldehyde groups into the cellulose molecular chain, providing reaction sites for subsequent covalent modification. Aminated silica and chitosan are premixed and then the oxidized leaf veins are modified. Under the heating condition of 50°C, on the one hand, the amino The amino groups of silica can form Schiff base bonds with the aldehyde groups on the leaf veins, achieving stable anchoring. On the other hand, chitosan molecular chains interact with silica nanoparticles and form multiple physicochemical crosslinks with the leaf veins and silica, ultimately constructing a uniform, stable, and functional composite coating rich in hydrophilic groups such as amino and hydroxyl groups on the surface and within the pores of the leaf vein framework. This composite coating significantly improves the material's hydrophilicity, cell affinity, and long-term culture stability. This multilayer covalent modification strategy greatly enhances the coating's adhesion and functionality.

[0024] (3) Excellent biocompatibility and clear proliferation-promoting effect: CCK-8 cytotoxicity assay and live / dead cell double staining assay confirmed that the scaffold prepared in this invention is non-toxic to mesenchymal stem cells and PC12 cells, exhibiting excellent biocompatibility. Cells can adhere well, spread out and proliferate in the three-dimensional space inside the scaffold, forming three-dimensional cell clusters, which have application potential in neural tissue engineering research and in vitro model construction. EdU cell proliferation experiment results showed that the proliferation activity of PC12 cells on the scaffold of this invention was significantly higher than that of conventional two-dimensional culture, proving that the scaffold can not only support cell survival, but also actively create a microenvironment conducive to cell proliferation.

[0025] (4) Raw materials are readily available, the process is green and simple, and it is easy to promote: All raw materials for this invention are widely available and inexpensive. The preparation process does not involve complex equipment or harsh conditions, the steps are simple, the reaction time is short, the reproducibility is good, and it has good prospects for industrial application. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1 The image shows a comparison of scanning electron micrographs of the surface of leaf veins of different plants after alkali treatment; where A is the leaf of Osmanthus fragrans, B is the leaf of Nandina domestica, C is the leaf of Ligustrum lucidum, and D is the leaf of Photinia serratifolia.

[0028] Figure 2 The image shows a multi-scale scanning electron microscope (SEM) image of a three-dimensional cellular scaffold (ND-SiO2-CS) based on Nandina domestica leaf veins obtained in Example 1 of this invention; where A is the complete leaf vein network; BC are the porous structures at different magnifications, showing pore sizes of 50-200 μm; D is the fiber skeleton of a single leaf vein; and EF are the high-magnification morphology of the fiber surface after modification with an amino-modified silica / chitosan composite coating. Detailed Implementation

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] Main material sources:

[0031] Leaves of Nandina domestica, Ligustrum lucidum, Osmanthus fragrans, and Photinia were collected from campus or public green areas.

[0032] Chitosan (degree of deacetylation ≥95%, viscosity 100-200 mpa.s): purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0033] Aminated silica sol (2% solids content, 20-30nm particle size): purchased from Nanjing Xianfeng Nanomaterials Technology Co., Ltd. It can also be prepared by modification with KH-550.

[0034] Sodium periodate, sodium hydroxide, glacial acetic acid, etc., were all of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0035] Mesenchymal stem cells (MSCs) were purchased from Cyagen (Guangzhou) Biotechnology Co., Ltd. PC12 cells were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee.

[0036] The CCK-8 kit, the live / dead cell double staining kit (Calcein-AM / PI), and the EdU cell proliferation assay kit were all purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0037] Example 1

[0038] Preparation of a three-dimensional cell scaffold (ND-SiO2-CS) based on Nandina domestica leaf veins

[0039] Step 1: Screening and pretreatment of plant leaf veins

[0040] Fresh, healthy, and pest-free leaves from privet, osmanthus, photinia, and nandina were collected. The leaves were soaked in deionized water and the surface dust was gently brushed away with a soft brush. The complete vein network was carefully dissected using a scalpel or tweezers (mainly preserving the midrib and lateral veins, and removing the leaf parenchyma tissue), yielding four types of coarse leaf samples.

[0041] Immerse each of the four leaf veins in a beaker containing a 5% (w / v) NaOH aqueous solution, ensuring the veins are completely submerged. Place the beaker on a 100°C heating plate and boil for 6 minutes. During this process, the solution will gradually darken in color. After treatment, quickly discard the hot alkaline solution and rinse the leaf veins repeatedly with plenty of warm deionized water at approximately 60°C until the pH of the washing solution reaches approximately 7.0. Remove the leaf veins and dry them overnight in a 60°C oven.

[0042] Four types of leaf vein samples were dried and adhered to conductive adhesive. After gold sputtering, their surface microstructure was observed under a scanning electron microscope (SEM). The experimental results are as follows: Figure 1 As shown.

[0043] The results are as follows Figure 1 As shown, the Nandina domestica leaf veins exhibited the clearest and most regular interconnected porous structure after treatment, with pore size measurements concentrated between 50-200 μm, which was significantly better than the other three leaf veins. Therefore, Nandina domestica leaf veins were selected for subsequent experiments.

[0044] Step 2: Selective oxidation with sodium periodate

[0045] Accurately weigh sodium periodate and dissolve it in deionized water to prepare a 0.1% sodium periodate aqueous solution. Immerse the dried Nandina domestica leaf veins (screened and pretreated in step 1) in this solution, ensuring the veins are completely submerged. Wrap the container in aluminum foil and allow it to react in the dark at room temperature (approximately 25°C) for 10 hours. After the reaction, remove the leaf veins, rinse them thoroughly with plenty of deionized water, and then soak them in deionized water, changing the water every 30 minutes. Repeat this washing process several times until the final wash solution does not turn blue when tested with starch-potassium iodide paper (indicating no residual periodate ions). Remove the cleaned leaf veins, dry them, and obtain oxidized Nandina domestica leaf veins (denoted as O-ND) for later use.

[0046] Step 3: Aminated silica / chitosan composite modification

[0047] (1) Preparation of chitosan solution: Weigh 1.5g of chitosan powder and add it to 100mL of 1% glacial acetic acid aqueous solution. Stir magnetically overnight to dissolve it completely and obtain a 1.5% chitosan solution.

[0048] (2) Preparation of the modification mixture: Take commercially available aminated silica sol (2% solid content) and calculate based on a mass ratio of aminated silica (solid) to chitosan (solid) of 1:2. To prepare 100g of the modification mixture, 1.5g of chitosan solid is required, and the required mass of aminated silica solid is 0.75g. Take the mass of the 2% aminated silica sol as 0.75g / 2% = 37.5g. Slowly add 37.5g of aminated silica sol to 62.5g of 1.5% chitosan solution and stir magnetically for 30 minutes to mix evenly, thus obtaining the modification mixture.

[0049] (3) Composite modification reaction: The oxidized Nandina domestica leaf veins (O-ND) prepared in step 2 were immersed in the above modification mixture, ensuring that the leaf veins were completely submerged. The entire container was placed in a constant temperature drying oven at 50°C and allowed to stand for 1.5 hours. After the reaction, the leaf vein scaffold was removed and gently rinsed three times with sterile phosphate-buffered saline (PBS, pH 7.4) for 5 minutes each time to remove physically adsorbed but not firmly bound components. The washed scaffold was pre-frozen in a -20°C freezer for 2 hours, and then freeze-dried in a freeze dryer for 24 hours to obtain the composite leaf vein three-dimensional cell scaffold, denoted as ND-SiO2-CS. The scaffold was sealed and packaged, sterilized by cobalt-60 irradiation (dose 15 kGy), and stored in a freezer at 4°C for later use.

[0050] The prepared ND-SiO2-CS scaffold was observed by SEM, and the results are as follows: Figure 2 As shown, the scaffold completely preserves the natural three-dimensional network framework of Nandina domestica leaf veins, with clearly visible and interconnected pores, indicating that subsequent chemical treatment did not damage its macroscopic structure. The surface of the leaf vein framework is uniformly covered with a composite coating composed of nano-sized particles. This coating does not block the pores of the leaf veins but rather tightly wraps around the surface of the fibrous framework.

[0051] Example 2

[0052] Effect of different sodium periodate concentrations on stent performance

[0053] This embodiment aims to investigate the effect of sodium periodate concentration in step 2 on the subsequent composite modification effect and the cell compatibility of the final scaffold. Except for the mass fraction of sodium periodate solution in step 2 being set to 0.01%, 0.1%, 0.5%, and 1%, the other preparation steps were exactly the same as in Example 1. The resulting scaffolds were named ND-SiO2-CS-0.01, ND-SiO2-CS-0.1, ND-SiO2-CS-0.5, and ND-SiO2-CS-1, respectively.

[0054] Semi-quantitative detection of surface aldehyde content: Schiff base colorimetric method was used. Oxidized leaf veins (O-ND) were reacted with 0.1% (w / v) methylene blue solution (the amino group of methylene blue can combine with the aldehyde group) at 37°C for 1 hour. After thorough washing, the color intensity was observed.

[0055] Table 1. Semi-quantitative detection results of aldehyde content in leaf veins after treatment with different sodium periodate concentrations.

[0056]

[0057] Note: The more "+" signs there are for staining depth, the darker the color and the higher the aldehyde content.

[0058] The results showed that as the sodium periodate concentration increased from 0.01% to 0.5%, the staining of leaf veins gradually deepened, indicating an increase in the amount of aldehyde groups introduced. When the concentration reached 1%, the staining depth did not increase significantly compared to the 0.5% group, indicating that the amount of aldehyde groups introduced was approaching saturation. The texture of the leaf veins changed slightly, but the overall three-dimensional structure remained intact. Considering both the efficiency of aldehyde group introduction and the integrity of the skeletal structure, a sodium periodate concentration of 0.1%–0.5% was optimal, as this range effectively introduced sufficient aldehyde groups while maintaining the integrity of the leaf vein structure.

[0059] Cell adhesion and proliferation assay: Four sterilized scaffolds were placed in 48-well plates, and PC12 cells were seeded (5 × 10⁶ cells per well). 4 (Number of cells). After culturing for 24 hours, unattached cells were gently washed away with PBS, and the attached cells were detected using the CCK-8 assay. After culturing for 72 hours, cell proliferation was again detected using the CCK-8 assay.

[0060] Table 2. Effects of scaffolds prepared with different sodium periodate concentrations on PC12 cell adhesion and proliferation (OD). 450 (mean ± standard deviation, n=5)

[0061] *Note: Multiplication rate = 72h OD value / 24h OD value*

[0062] Compared with the 0.01% group, p < 0.05 (24h) or p < 0.01 (72h)

[0063] Compared with the 0.5% group, p < 0.05

[0064] Compared with the 0.1% and 0.5% groups, p < 0.01

[0065] In summary, both 0.1% and 0.5% concentrations effectively introduce aldehyde groups, providing sufficient reaction sites for subsequent modifications. Considering that a 0.1% concentration already achieves good results and causes less damage to the skeleton, 0.1% is selected as the preferred concentration in subsequent examples.

[0066] Example 3

[0067] Effect of different aminated silica / chitosan ratios on scaffold performance

[0068] This embodiment aims to investigate the effect of the mass ratio of aminated silica to chitosan in step 3 on the performance of the final scaffold. The chitosan solution concentration was kept constant at 1.5%, and the mass ratio of aminated silica (solid) to chitosan (solid) was varied to 1:1, 1:2, and 1:4, respectively. All other steps were identical to those in Example 1. The resulting scaffolds were named ND-SiO2-CS-1:1, ND-SiO2-CS-1:2, and ND-SiO2-CS-1:4, respectively.

[0069] Coating stability test: The scaffold was immersed in PBS and shaken at 37°C (100 rpm) for 7 days. The supernatant was collected on days 1, 3, and 7, and the protein / polysaccharide content (representing detached chitosan) in the supernatant was determined by the Coomassie Brilliant Blue method, and the silicon content (representing detached silica) in the supernatant was determined by ICP-MS. The results are shown in Table 3.

[0070] Table 3. Coating detachment of different scaffold ratios after shaking culture in PBS (mean ± standard deviation, n=3)

[0071] The results showed that in the initial stage (1 day), the ND-SiO2-CS-1:1 group had a lot of silica and chitosan detached, possibly due to the high proportion and physical adsorption as the main process; the ND-SiO2-CS-1:4 group had a small amount of chitosan detached throughout the entire cycle; while the ND-SiO2-CS-1:2 group had the least amount of coating detachment and was the most stable throughout the entire cycle.

[0072] Cell viability evaluation: Three sterilized scaffolds were seeded into PC12 cells. After 3 days of culture, live / dead cell staining was performed, and the fluorescence images were quantitatively analyzed using ImageJ software to calculate cell viability, cell spreading area, and cell cluster number. The results are shown in Table 4.

[0073] Table 4. Viability and morphological parameters of PC12 cells on scaffolds with different ratios (mean ± standard deviation, n=5)

[0074] The results showed that cells survived well on all three scaffolds, but the ND-SiO2-CS-1:2 group had a larger cell spreading area and formed more cell clusters. Based on the stability test results, the optimal ratio was 1:2.

[0075] Example 4

[0076] Biocompatibility evaluation of scaffolds (CCK-8 assay)

[0077] In this embodiment, the CCK-8 assay was used to evaluate the cytotoxicity of the scaffold prepared in Example 1 of the present invention to mesenchymal stem cells (MSCs).

[0078] Experimental Groups:

[0079] Experimental group: ND-SiO2-CS scaffold prepared in Example 1.

[0080] Positive control group: Cells seeded on a standard tissue culture plate (TCP).

[0081] Blank control group: only culture medium, no cells.

[0082] Experimental steps:

[0083] (1) Carefully place the sterilized ND-SiO2-CS scaffold into the 96-well plate, one scaffold per well.

[0084] (2) Take third-generation mouse mesenchymal stem cells in the logarithmic growth phase, digest them with 0.25% trypsin, count them, resuspend them in DMEM medium containing 10% fetal bovine serum, and adjust the cell density to 1×10⁻⁶. 5 per mL.

[0085] (3) Add 100 μL of cell suspension (i.e., 1×10⁶ cells per well) to the wells of the experimental group and the positive control group, respectively. 4 (100 cells). Add 100 μL of cell-free culture medium to the wells of the blank control group. Set up 5 replicates for each group.

[0086] (4) Place the culture plates in an incubator at 37°C and 5% CO2 and incubate for 1 day, 4 days and 7 days respectively. Replace with fresh culture medium every 2 days.

[0087] (5) At each detection time point, discard the old culture medium and rinse gently once with PBS. Add 100 μL of complete culture medium containing 10% CCK-8 reagent to each well and continue incubation for 2 hours.

[0088] (6) After incubation, transfer 100 μL of supernatant from each well to a new 96-well plate and measure the absorbance (OD) at 450 nm using a microplate reader. 450 ).

[0089] Table 5. CCK-8 assay results for cells in different culture groups at different times.

[0090] (OD) 450 (mean ± standard deviation, n=5)

[0091] *Note: Data are expressed as mean ± standard deviation (n=5). There were no significant differences between the experimental group and the positive control group at any time point (p > 0.05).

[0092] Results analysis: As shown in Table 5, after 1 day, 4 days, and 7 days of culture:

[0093] Compared with the positive control group, the OD of hMSCs seeded on ND-SiO2-CS scaffolds was higher in the experimental group. 450 The values ​​were not significantly different from those of the TCP control group (p > 0.05), indicating that the scaffold was not cytotoxic to hMSCs.

[0094] Cell proliferation trend: The OD values ​​of both groups increased steadily with the extension of culture time, indicating that cells can continuously proliferate on the scaffold, and the scaffold provides a suitable three-dimensional microenvironment for cell growth.

[0095] Blank control group: OD values ​​at all time points were below 0.07, indicating that the culture medium itself did not interfere with the test results.

[0096] This result fully demonstrates that the ND-SiO2-CS scaffold prepared in this invention is non-cytotoxic to hMSCs and has excellent biocompatibility.

[0097] Example 5

[0098] Evaluation of cell viability status of scaffolds (live / dead cell staining)

[0099] This embodiment uses the live / dead cell staining method to visually assess the cell viability on the scaffold.

[0100] Experimental steps:

[0101] (1) Place the sterilized ND-SiO2-CS scaffold into a 24-well plate.

[0102] (2) Take PC12 cells in the logarithmic growth phase, digest and count them, resuspend them in DMEM-F12 medium containing 10% fetal bovine serum, and adjust the cell density to 2×10⁶ cells / year. 5 Cells / mL. Add 500 μL of cell suspension to each well (i.e., 1 × 10⁶ cells / mL). 5 (cells).

[0103] (3) Place the culture plate in an incubator at 37°C and 5% CO2 for 5 days, and change the culture medium every 2 days during the period.

[0104] (4) After the culture is completed, the culture medium is discarded and the scaffold is gently rinsed twice with PBS preheated to 37°C.

[0105] (5) Prepare live / dead cell staining working solution with fresh PBS, so that the final concentration of Calcein-AM is 2 μM and the final concentration of PI is 2 μM.

[0106] (6) Add 500 μL of staining working solution to each well, ensuring the scaffold is completely submerged. Incubate in a 37°C incubator for 30 minutes, protected from light.

[0107] (7) After incubation, aspirate the staining solution and rinse once with PBS. Immediately place the support under a laser confocal microscope for observation and photography. Randomly select 5 non-overlapping fields of view for image acquisition in each group, and use ImageJ software to perform quantitative analysis of the fluorescence images.

[0108] Table 6. Quantitative analysis of live / dead staining of PC12 cells after 5 days of culture on ND-SiO2-CS scaffolds (mean ± standard deviation, n=5)

[0109] Results Analysis: Cell Viability: Quantitative analysis showed that after 5 days of culture on the ND-SiO2-CS scaffold, the cell viability of PC12 cells was as high as 97.7 ± 1.8%, indicating that the scaffold has excellent cell compatibility and no toxic effect on PC12 cells.

[0110] Cell distribution and morphology: Cells are evenly distributed on the scaffold, arranged orderly along the leaf vein fiber skeleton, and aggregate in the scaffold pores to form three-dimensional cell clusters. Most cells exhibit a spindle-shaped or multi-protrusion-like extended morphology, indicating that the cells can spread and grow normally on the scaffold.

[0111] Three-dimensional growth characteristics: an average of 18.5 cell clusters were visible per field of view, with an average cluster area of ​​2850.6 μm², indicating that cells not only grow on the surface of the scaffold, but also migrate into the pores inside the scaffold to form a three-dimensional spatial distribution, thus successfully realizing three-dimensional cell culture.

[0112] Example 6

[0113] Evaluation of the cell proliferation-promoting capacity of scaffolds (EdU method)

[0114] This embodiment uses the EdU incorporation method to quantitatively evaluate the effect of the scaffold on the proliferation capacity of PC12 cells.

[0115] Experimental group: Experimental group: ND-SiO2-CS scaffold.

[0116] Two-dimensional control group: seeded in 24-well plates containing cell spreaders.

[0117] Experimental steps:

[0118] (1) Place the sterilized scaffold and cell spreader into a 24-well plate respectively.

[0119] (2) After digestion, the density of PC12 cells was adjusted to 1×10⁻⁶. 5 Cells / mL. Add 500 μL of cell suspension (5 × 10⁶ cells / mL) to each well. 4 (1 cell), with 3 replicates per group.

[0120] (3) Culture for 24 hours to allow the cells to adhere to the scaffold or climbing slide.

[0121] (4) EdU labeling: Dilute the EdU stock solution with complete culture medium to prepare 2× EdU working solution (final concentration of 20 μM). Discard the old culture medium in each well, add 500 μL of fresh complete culture medium containing 10 μM EdU, and continue to incubate for 24 hours.

[0122] (5) Cell fixation and permeabilization: Discard the culture medium, add 500 μL of 4% paraformaldehyde to each well, and fix at room temperature for 30 minutes. Discard the fixative, add 500 μL of 2 mg / mL glycine solution to each well, and incubate for 5 minutes. After rinsing with PBS, add 500 μL of PBS containing 0.5% Triton X-100 to each well, and permeabilize at room temperature for 10 minutes.

[0123] (6) Click-iT reaction: Prepare the Click-iT reaction solution (containing Alexa Fluor 488 azide) according to the kit instructions. Add 350 μL of the reaction solution to each well and incubate at room temperature in the dark for 30 minutes. Discard the reaction solution and wash with PBS containing 0.5% Triton X-100.

[0124] (7) Nuclear staining: Prepare Hoechst 33342 working solution (5 μg / mL) with PBS. Add 350 μL of Hoechst staining solution to each well and incubate at room temperature in the dark for 30 minutes. Rinse with PBS.

[0125] (8) Mounting and observation: Carefully remove the support and slide, place them on a glass slide, add anti-fluorescence quenching mounting medium, and cover with a coverslip. Take pictures of at least 5 non-repeating fields of view under a fluorescence microscope. Hoechst 33342 stains all cell nuclei, showing blue fluorescence; EdU-positive cell nuclei show green fluorescence.

[0126] (9) Counting and analysis: The total number of blue cell nuclei and green cell nuclei in each photo were counted using Image-Pro Plus software, and then the cell proliferation rate was calculated as (number of EdU positive cells / total number of Hoechst-stained cells) × 100%.

[0127] Table 7. Results of EdU assay for PC12 cell proliferation on scaffolds and two-dimensional control groups.

[0128]

[0129] *Note: Relative proliferation rate = Average proliferation rate of experimental group / Average proliferation rate of two-dimensional control group

[0130] Results Analysis: The EdU positivity rate of PC12 cells cultured on the ND-SiO2-CS scaffold (38.0 ± 3.1%) was significantly higher than that of the two-dimensional scaffold control group (20.0 ± 2.4%), p < 0.01. The cell proliferation rate of the scaffold group was approximately 1.9 times that of the two-dimensional control group, quantitatively demonstrating that the scaffold prepared in this invention can effectively promote DNA synthesis and cell proliferation of PC12 cells.

[0131] Example 7

[0132] Application of scaffolds in different cell lines

[0133] This embodiment applies the scaffold of the present invention to the culture of other cell types to verify its broad applicability. In addition to the MSC and PC12 cells mentioned above, this embodiment attempts to culture human skin fibroblasts (HFF-1) and human liver cancer cells (HepG2).

[0134] The experimental procedures were the same as in Examples 4 and 5. After 7 days of culture, the morphology of the cells on the scaffold was observed by SEM, and cell viability was detected by CCK-8 assay.

[0135] The results showed that HFF-1 cells extended well on the scaffold, exhibiting a typical elongated spindle shape, and secreted a large amount of extracellular matrix to encapsulate themselves. HepG2 cells, on the other hand, aggregated in the pores of the scaffold, forming a three-dimensional structure resembling hepatocyte spheres, and CCK-8 results showed good activity. This indicates that the scaffold prepared in this invention has good broad applicability and can support the three-dimensional growth of different cell types.

[0136] In summary, this invention provides a three-dimensional cell scaffold based on Nandina domestica leaf veins, its preparation method, and its applications. The scaffold uses Nandina domestica leaf veins with ideal pore sizes (50-200 μm) and interconnected structures as the substrate. A stable composite functional coating formed by aminated silica and chitosan is firmly constructed on the surface of the leaf vein skeleton through a three-step process of "alkali boiling-oxidation-composite modification." This preparation method is simple, mild, environmentally friendly, and easily scalable. The resulting scaffold exhibits excellent biocompatibility, is non-toxic to various cell types, and significantly promotes cell adhesion, spreading, and proliferation. Therefore, the three-dimensional cell scaffold provided by this invention has broad industrial application prospects in biomedical fields such as tissue engineering, regenerative medicine, drug screening, and in vitro disease model construction.

[0137] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0138] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a three-dimensional cell scaffold based on Nandina domestica leaf veins, characterized in that, Includes the following steps: S1： Pretreatment: Select the veins of Nandina domestica leaves, immerse them in a 5% sodium hydroxide solution, boil at 100℃ for 5-7 minutes for pretreatment, then remove, wash and dry; S2: Selective oxidation: The pretreated Nandina domestica leaf veins were immersed in sodium periodate solution for selective oxidation, and then washed and dried after the reaction. S3: Composite modification: Aminated silica was mixed with a chitosan solution with a mass fraction of 1.5% to obtain a mixture with a mass ratio of 1:2 between the solid aminated silica and the solid chitosan. The oxidized Nandina domestica leaf veins were then immersed in the mixture and reacted at 50°C for 1.5 hours. After removal, washing, and freeze-drying, a composite leaf vein three-dimensional cell scaffold was obtained.

2. The method according to claim 1, characterized in that, After pretreatment, the veins of Nandina domestica leaves described in step S1 exhibit a porous network with a pore size of 50–200 μm and a uniform structure.

3. The method according to claim 1, characterized in that, The sodium periodate solution in step S2 has a mass fraction of 0.01% to 1%; the selective oxidation conditions are as follows: reaction at room temperature in the dark for 6 to 12 hours.

4. The method according to claim 1, characterized in that, The chitosan solution in step S3 is prepared using a 1% (v / v) aqueous solution of glacial acetic acid as a solvent; the aminated silica is added in sol form, with a solid content of 1% to 3%.

5. The method according to claim 1, characterized in that, The sodium periodate solution in step S2 has a mass fraction of 0.1% to 0.5%.

6. A three-dimensional cell scaffold based on Nandina domestica leaf veins, characterized in that, It is prepared by the method described in any one of claims 1 to 5.

7. The application of the three-dimensional cell scaffold according to claim 6 in three-dimensional cell culture.

8. The application according to claim 7, characterized in that, The cells include mesenchymal stem cells or PC12 cells.

9. The application according to claim 7, characterized in that, The scaffold demonstrated good biocompatibility through CCK-8 assay for cell viability and live / dead cell double staining experiments, and its ability to promote cell proliferation was demonstrated through EdU cell proliferation assay.

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