Multifunctional collagen bio-ink for DLP (Digital Light Processing) 3D (Three-Dimensional) printing as well as preparation method and application of multifunctional collagen bio-ink

Abstract

The invention relates to the technical field of biomedical materials, in particular to multifunctional collagen bio-ink for DLP 3D printing as well as a preparation method and application thereof.The bio-ink is prepared from components as follows: 0.5%-1.5% m / v of a methacrylated collagen solution, 0.125%-0.5% m / v of a photoinitiator, 0.025%-0.1% m / v of an ultraviolet absorbent and 0.05 mg / mL-0.4 mg / mL of a dihydromyricetin solution, the hydrogel has the advantages of good fluidity, high gelation modulus, accurate cell-carrying printability and high biocompatibility. The gel has excellent oxidation resistance, a 3D culture environment without oxidative stress is provided for HDFs, proliferation and spreading of HDFs on the gel are remarkably promoted, and the biological activity of the gel is improved; the artificial skin printed by utilizing the bio-ink loaded HDFs has an excellent treatment effect on diabetic wounds, and has good clinical application potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials and relates to a multifunctional collagen bio-ink for DLP 3D printing, a preparation method and an application thereof. Background Art

[0002] Diabetes is an increasingly serious global epidemic that has a serious impact on the quality of life of patients and the consumption of medical resources. Diabetes is prone to induce complications, such as diabetic nephropathy, diabetic foot, diabetic retinopathy, and diabetic heart disease. Diabetic complications are irreversible and once they occur, patients will carry them for life. At the same time, the complex effects of hyperglycemia, including increased oxidative stress levels, persistent inflammatory responses, and damage to the vascular nervous system, hinder wound healing in diabetic patients. The treatment of diabetic wounds requires comprehensive consideration of multiple factors, and comprehensive measures such as antioxidant therapy, oxygen therapy, blood sugar control, and bioactive substances are taken to promote wound healing, reduce the risk of complications, and improve the quality of life of patients. However, the above treatment methods also have some limitations, such as oxygen therapy is limited by microvascular lesions, blood sugar control is difficult, and the selection and dosage of bioactive substances need to be carefully considered. Therefore, new treatment methods / materials need to be developed to better treat diabetic wounds. 3D bioprinting technology is one of the most advanced technologies in the field of tissue engineering. It can use biocompatible materials and cells to construct complex biological tissues and organ structures layer by layer, such as artificial skin, bone and cartilage materials, and is expected to become a new material for treating diabetic wounds.

[0003] 3D printing technology can be divided into many types according to the working principle and materials, including FDM, SLA, SLS and DLP. These technologies have the characteristics of manufacturing flexibility, rapid prototyping, and high resource utilization efficiency. Among them, DLP 3D printing is known for its high speed, high precision and good surface smoothness, and is particularly suitable for application scenarios that require high precision and high speed. The key to DLP 3D printing is to prepare ideal bio-ink. The ideal DLP bio-ink should have multiple properties to meet the needs of different types of bio-printing: (1) Good fluidity is essential to ensure that the bio-ink flows evenly during the printing process, thereby achieving high-precision printing; (2) The bio-ink should have good cell-loading biocompatibility to ensure that the printed structure can interact well with cells and promote cell growth and differentiation; (3) The printed gel or structure should also have appropriate strength and structural stability to ensure that it can provide sufficient support and stability during implantation or application, thereby promoting tissue regeneration and healing.

[0004] As an important protein in the human body, collagen has multiple advantages in 3D printing applications. For example, it has good biocompatibility and bioactivity, and is not prone to rejection reactions; it is easy to form a gel, which is beneficial to shape stability during printing and helps the growth of cells in the gel. However, collagen has low solubility, poor structural stability, and is prone to shrinkage and deformation, which limits its application in 3D printing. Therefore, how to improve the mechanical properties of collagen and thus improve its stability has become a technical problem that needs to be solved urgently by those skilled in the art.

[0005] Dihydromyricetin (DHM), also known as dihydromyricetin and ampelopsis, is a flavonol compound extracted from the Chinese herbal medicine Ampelopsis serrulata, which is widely distributed in southern China. In previous studies, dihydromyricetin has hepatoprotective, antioxidant, cardioprotective, anti-inflammatory and hypoglycemic effects. Studies have shown that it can inhibit the proliferation of liver cancer cells, breast cancer cells, prostate cancer cells and bladder cancer cells, and promote apoptosis of various tumor cells. The mechanism of action is related to inducing apoptosis of tumor cells, blocking the cell cycle of tumor cells, inducing autophagy of tumor cells, and inhibiting the invasion of tumor cells.

[0006] The inventor unexpectedly discovered during the research process that dihydromyricetin can be used as a cross-linking agent for collagen, and the prepared bio-ink has good fluidity, higher gelation modulus, lower swelling ratio, and better anti-degradation performance; it can print three-dimensional complex models, maintain micron-level printing accuracy while having higher cell-carrying biocompatibility and structural stability, and can promote cell proliferation in the printed gel, showing its potential as a tissue engineering bio-ink; the printed gel has excellent antioxidant properties, can effectively remove reactive oxygen species (ROS), and provide a 3D culture environment without oxidative stress for human fibroblasts (HDFs), significantly promoting the proliferation and spreading of HDFs on the gel, and further improving the biological activity of the collagen gel; the artificial skin prepared by the bio-ink loaded with human fibroblasts (HDFs), after 14 days of culture, the gel still maintains good structural integrity, and the cells in the gel proliferate significantly, with a very high cell survival rate; it is applied to the treatment of chronic diabetic wounds, showing faster healing speed and better healing quality, highlighting the potential advantages of cell-carrying artificial skin in the treatment of diabetic wounds, and has good clinical application potential. Summary of the invention

[0007] The object of the present invention is to provide a multifunctional collagen bio-ink for DLP 3D printing, wherein the bio-ink comprises the following components: 0.5%-1.5% m / v methacryloyl collagen solution, 0.125%-0.5% m / v photoinitiator, 0.025%-0.1% m / v UV absorber and 0.05-0.4 mg / mL dihydromyricetin solution.

[0008] Preferably, the collagen is bovine collagen.

[0009] Preferably, the bovine collagen is type I bovine collagen, and / or type II bovine collagen, and / or type II bovine collagen.

[0010] Preferably, the photoinitiator includes one or more of LAP, TPO, and I2959.

[0011] Preferably, the photoinitiator is LAP.

[0012] Preferably, the ultraviolet absorber includes lemon yellow and / or food coloring brilliant blue.

[0013] Preferably, the ultraviolet absorber is lemon yellow.

[0014] The second object of the present invention is to provide a method for preparing the bio-ink, comprising the following steps:

[0015] (1) Dissolve the freeze-dried collagen sponge in acetic acid solution and stir at low temperature until it is completely dissolved;

[0016] (2) adding methacrylic anhydride dropwise to the collagen solution obtained in step (1), adjusting the pH to 4-9, reacting at low temperature for 24-48 hours, dialyzing, and freeze-drying to obtain methacrylylated collagen;

[0017] (3) The methacryloyl collagen obtained in step (2) is dissolved in acetic acid solution to a concentration of 0.5%-1.5% m / v, and sodium hydroxide solution is added to adjust the pH of the solution to neutral; 0.125%-0.5% m / v photoinitiator, 0.025%-0.1% m / v ultraviolet absorber and 0.05-0.4 mg / mL dihydromyricetin solution are added, mixed evenly, and centrifuged to remove bubbles to obtain CMA-DHM bio-ink.

[0018] The third object of the present invention is to provide one or more applications of the biological ink in the preparation of tissue engineering materials, hemostatic materials, wound dressings, and drug delivery materials.

[0019] Preferably, the tissue engineering material comprises one or more of artificial skin, artificial blood vessels, cartilage and bone repair materials and corneal transplant materials.

[0020] The fourth object of the present invention is to provide an artificial skin for treating chronic diabetic wounds, wherein the artificial skin is obtained by adding human fibroblasts to the biological ink and printing it through DLP 3D.

[0021] The fifth object of the present invention is to provide the use of the artificial skin in treating diabetic chronic wounds.

[0022] The present invention has the following beneficial effects:

[0023] (1) The present invention provides a multifunctional collagen bio-ink for DLP 3D printing, wherein the bio-ink comprises the following components: 0.5%-1.5% m / v methacryloyl collagen solution, 0.125%-0.5% m / v photoinitiator, 0.025%-0.1% m / v UV absorber and 0.05-0.4 mg / mL dihydromyricetin solution, wherein the bio-ink has good fluidity, higher gelation modulus, lower swelling ratio and better anti-degradation performance; it can print three-dimensional complex models, maintain micron-level printing accuracy while having higher cell-carrying biocompatibility and structural stability, can promote cell proliferation in the printed gel, and shows the potential as a tissue engineering bio-ink;

[0024] (2) The bio-ink printed gel of the present invention has excellent antioxidant properties, can effectively remove reactive oxygen species (ROS), provide a 3D culture environment without oxidative stress for human fibroblasts (HDFs), significantly promote the proliferation and spreading of HDFs on the gel, and further improve the biological activity of the collagen gel;

[0025] (3) The artificial skin prepared by the bio-ink loaded with human fibroblasts (HDFs) described in the present invention maintained good structural integrity after 14 days of culture, and the cells in the gel proliferated significantly with a very high cell survival rate. It was applied to the treatment of chronic diabetic wounds and showed faster healing speed and better healing quality, highlighting the potential advantages of cell-loaded artificial skin in the treatment of diabetic wounds and having good clinical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1Screening of DHM concentration Note: a. Skeleton staining of L929 cells grown on the surface of CMA, CMA-DHM (0.1), CMA-DHM (0.2), and CMA-DHM (0.4) gels; b. DCFH-DA staining of L929 cells grown on the surface of CMA, CMA-DHM (0.1), CMA-DHM (0.2), and CMA-DHM (0.4) gels after treatment with hydrogen peroxide; c. Live and dead cell staining of L929 cells encapsulated with CMA, CMA-DHM (0.1), CMA-DHM (0.2), and CMA-DHM (0.4) bio-inks for 4 days.

[0027] Figure 2 Study on the printability of CMA-DHM Note: a. In-situ curing rheological characterization of CMA and CMA-DHM; b. Circular dichroism characterization of CMA and CMA-DHM inks; c. Curing time of inks with different LAP concentrations; d. Curing time of inks with different UA concentrations; e. CAD comb model; f. Comb model after printing; g. Rose model and multi-angle photos after printing; h. Three-dimensional hollow model and multi-angle photos after printing; i. Photos of the three-dimensional hollow model before and after compression; j. Grid photos after printing; k. SEM characterization of the gel surface and cross section after printing. Figure 3 Cross-linking mechanism of CMA-DHM and characterization of gel properties Note: a. Infrared spectra of CMA and CMA-DHM; b. Characterization of storage modulus and loss modulus of CMA and CMA-DHM gels at different strains; c. Dynamic viscosity of CMA and CMA-DHM gels at different shear rates; d. Stress-strain curves of CMA and CMA-DHM gels; e. Swelling rate of CMA and CMA-DHM gels; f. Mass residual rate of CMA and CMA-DHM gels in collagenase; g. Schematic diagram of non-covalent cross-linking of DHM to CMA; h. Schematic diagram of covalent cross-linking of DHM to CMA.

[0028] Figure 4Biocompatibility of CMA-DHM cells Note: a. Live and dead cell staining images of L929 cells encapsulated with CMA and CMA-DHM bio-inks after 1, 4, and 7 days of culture; b. Live and dead cell counts of L929 cells encapsulated with CMA and CMA-DHM bio-inks after 1, 4, and 7 days of culture; c. Live cell quantification of L929 cells encapsulated with CMA and CMA-DHM bio-inks through CCK 8 experiment after 1, 4, and 7 days of culture; d. Live and dead cell staining images of CMA-DHM bio-ink-loaded cell printing after 1, 4, and 7 days of culture; e. Live cell counts of CMA-DHM bio-ink-loaded cell printing after 1, 4, and 7 days of culture; f. Bright field image of CMA-DHM bio-ink-loaded cell printing after 1 day of culture; g. Gray value of the green line in Figure f analyzed by Image J; h. Average width of the grid side length of CMA-DHM bio-ink-loaded cell printing after 1, 4, and 7 days of culture.

[0029] Figure 5 Antioxidant properties of CMA-DHM gel Note: a. Schematic diagram of the antioxidant properties of CMA-DHM gel; b. Quantification of HDFs cells grown on CMA and CMA-DHM gels for 3 days; c. Cytoskeleton staining of HDFs grown on CMA and CMA-DHM gels for 3 days; d. Cellular reactive oxygen levels and live-dead cell staining on Control, CMA and CMA-DHM gels stained with DCFH-DA; e. Fluorescence semi-quantitative analysis of DCFH-DA staining; f. Fluorescence intensity of reactive oxygen species stained with DCFH-DA in Control, CMA and CMA-DHM samples; g. Relative content of dead cells in Control, CMA and CMA-DHM samples.

[0030] Figure 6 Study on diabetic wound healing of cell-laden artificial skin Note: a. Design of artificial skin model and in vitro culture live and dead cell staining; b. Wound appearance at different time points of Control, CMA, CMA-DHM, CMA-DHM@HDFs and acute wound control group; c. Wound healing overlay processed by Image J; d. Quantification of wound closure rate. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings of the specification. Obviously, the described embodiments are only a part of the present invention, not the whole of the invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] In the following examples, unless otherwise specified, the methods used are all conventional methods;

[0033] In the following examples, unless otherwise specified, the reagents used are conventional reagents and can be purchased from the market.

[0034] In the following examples, unless otherwise specified, the reagents used in the process of preparing the bio-ink are not limited to specific concentrations, are not critical to the preparation, or can be achieved at any concentration.

[0035] In the embodiment of the present invention, yak type I collagen is used as raw material (for the extraction method, see invention patent CN113520900B) for DLP 3D printing collagen bio-ink, but the present invention is not limited to bovine collagen, and collagen obtained by any other means may be used, including type I collagen, type II collagen, type III collagen, etc.

[0036] In the following embodiments, the collagen DLP 3D printing bio-ink is called bio-ink before printing and is called hydrogel after DLP 3D printing.

[0037] Example 1 Preparation and formulation screening of CMA-DHM ink

[0038] Dissolve the freeze-dried collagen sponge in acetic acid solution and stir at low temperature until completely dissolved; add methacrylic anhydride to the dissolved collagen solution and use NaOH solution to adjust the pH to maintain between 4 and 9. After low-temperature reaction for 24-48 hours, dialyze with ultrapure water and freeze-dry to obtain methacrylylated collagen (CMA).

[0039] According to the ink formula in Table 1, 12 kinds of bio-inks were prepared, in which the concentration of CMA solution was 0.5% to 1.5% m / v, the concentration of DHM was 0.05 to 0.4 mg / mL, the concentration of photoinitiator LAP was 0.125% to 0.5% m / v, and the concentration of UV absorber lemon yellow was 0.025% to 0.1% m / v. The bio-inks prepared within these ranges are in line with the mechanism of DLP 3D printing, so bio-inks not limited to these 12 formulas can be prepared according to different application requirements.

[0040] The effects of different DHM concentrations in bio-inks 1-4 on the ink system were studied. All four inks have good fluidity and meet the requirements of DLP 3D printing. Bio-ink 1 has little improvement on the mechanical properties of the ink and is not used in subsequent experiments; bio-inks 2-4 all have good printability. L929 fibroblasts were seeded on the surface of the gel scaffold printed with bio-inks 2-4, and CMA without DHM was used as a control. After 3 days of culture, phalloidin and Hoechst 33342 were used to stain the cytoskeleton and nucleus, respectively. The results are shown in Figure 2. Figure 1As shown in a, the number of cells proliferating on the gels prepared by bio-inks 2-4 was more than that on the CMA gel, and the number of cells on the gel prepared by bio-ink 2 was significantly more than that on the other groups. In addition, the L929 cells grown on the gel were treated with hydrogen peroxide for 40 minutes, and the DCFH-DA fluorescent probe was used to stain the oxidative stress level of the cells. The results are shown in Figure 1 As shown in b. The cells in the CMA group all showed oxidative stress under the stimulation of hydrogen peroxide, while the number of cells in the gel prepared by bio-ink 2-4 was extremely small due to the addition of antioxidant DHM, which was significantly less than that in the CMA group. Among them, the reactive oxygen fluorescence of the gel cells in bio-ink 2 was the weakest, indicating that this gel was most suitable for cell growth. At the same time, bio-ink 2-4 was used to encapsulate L929 cells, and live and dead cell staining was performed after 4 days of culture. The results showed that the cells in bio-ink 2 grew the most ( Figure 1 c). In summary, through cell experiments, it was found that the DHM concentration in bio-ink 2 was optimal.

[0041] To further optimize the bio-ink formulation, we increased the CMA concentration and prepared bio-ink 5-11. The optimal concentrations of the photoinitiator LAP and the UV absorber tartrazine in bio-ink 5-9 were studied, and the curing time was characterized by rheological experiments. The results are shown in Figure 2 c and 2d. Bio-ink 5 is not suitable due to the small amount of LAP added and the need to extend the curing time; Bio-ink 7 is not suitable due to the large amount of LAP added and the short curing time, but considering the high cytotoxicity of LAP. Bio-ink 8 is not suitable for printing complex three-dimensional models due to the small amount of UA added and the short curing time; Bio-ink 9 is not suitable due to the large amount of UA added and the long curing time. Bio-ink 6 is suitable for DLP 3D printing due to its suitable curing time and printing accuracy, and has become the preferred formula and used in subsequent cell-free three-dimensional model printing and gel experiments.

[0042] In the cell-carrying experiment, the concentration of LAP was appropriately reduced to prepare bio-ink 10, which was used in subsequent gel biofunctional experiments, cell encapsulation and cell-carrying printing experiments. When preparing animal experimental samples, in order to further improve the anti-degradation performance of the gel, the CMA concentration was further increased, and bio-ink 11 was used. Bio-ink 12 was prepared by further increasing the CMA concentration. Due to the high viscosity of the ink, it is not suitable for printing fine three-dimensional complex models, but only for printing simple two-dimensional models. This formula will not be used in subsequent experiments. Due to the large number of variables in the bio-ink formula, the flexibility of adjustment is extremely high. By adjusting the formula within the range listed in this article, a series of bio-inks with high biocompatibility and high-precision printing can be obtained.

[0043] Table 1 Formulations of different bio-inks

[0044]

[0045]

[0046] Example 2 Study on the printing performance of CMA-DHM ink

[0047] Rheological experiment: A parallel plate with a diameter of 15 mm and a portable curing light source (wavelength 405 nm, light intensity 25 mW / cm 2 ) was subjected to a 70 s time sweep oscillation test (strain γ = 1%, angular frequency ω = 1 rad / s, gap 0.7 mm) to determine the storage modulus (G') and loss modulus (G") of the in situ gelation of CMA and CMA-DHM inks.

[0048] Circular dichroism test: Circular dichroism characterization was performed on CMA and CMA-DHM solutions at a concentration of 1 mg / mL. The samples were equilibrated at 4 °C for at least 24 hours before CD measurement. A cuvette with a pathlength of 1 mm was used, and the wavelength scan range was 180 to 260 nm with a step size of 1.0 nm and an averaging time of 1.0 s.

[0049] DLP 3D printing: Comb models with different tooth spacing (1-5mm), rose models, three-dimensional hollow bracket models and 10×10 grid models were designed and printed. The printed comb models were photographed and recorded. In order to show the details of the printed models more clearly, the printed roses and three-dimensional hollow models were dyed blue or red for 5 seconds to assist in demonstrating the support performance of the printed gel. Blue dye was added to the grid model to further demonstrate its printing accuracy.

[0050] Scanning electron microscopy (SEM) characterization: After freeze-drying, the CMA-DHM printed gel was gold-sprayed, and the morphology of the gel surface and cross section was photographed under a scanning electron microscope.

[0051] The results are as follows Figure 2 As shown in Figure 2, compared with pure CMA, the ink after adding DHM showed a significantly higher storage modulus under the same blue light irradiation time, indicating that the CMA-DHM ink has a higher viscoelasticity ( Figure 2 a). At the same time, the CMA-DHM ink maintains a complete triple helix structure ( Figure 2 b). By screening different LAP and UA concentrations ( Figure 2 cd) and determined the best ink formula, and carried out printing experiments on different models. For the comb tooth models with different tooth spacing (1-5mm), the printing effect is relatively ideal, and the comb teeth with the smallest spacing can also be clearly distinguished ( Figure 2f), indicating the applicability of CMA-DHM ink in DLP 3D printing. A rose model with a height of 1.5 cm was printed using CMA-DHM ink ( Figure 2 g), the details of the printed petals (i), the hollow part of the flower (ii), and the edge of a single petal (iii) are clearly visible, demonstrating the high accuracy of the printing. The printed flower model can stand stably when placed on a finger (iv) or a gun tip (v), indicating that the printed gel has sufficient support.

[0052] The hollow three-dimensional model was further printed ( Figure 2 h), where the mesh is clearly visible (ii). A transparent tube of the same size as the mesh passed through the printed hollow gel without any resistance, indicating that the printed mesh was not adhered (iii) and the gel had good support (iv and v). To verify the support of the printed gel, we applied pressure to it for 10 seconds and then released it immediately. We found that the printed gel did not break and returned to its original shape ( Figure 2 i), indicating that the printed gel has a certain mechanical strength and can withstand a certain amount of stress. The 10×10 grid model further demonstrates the high-resolution printing effect ( Figure 2 j). The edges of 100 mesh holes are clearly visible. After adding dye, the distinction between mesh holes and edges is more obvious, and the printing quality is uniform (iii). SEM characterization found that the surface and cross section of the printed gel showed a uniform porous structure ( Figure 2 k). Together, these experimental results indicate that CMA-DHM ink has excellent rheological properties, structural stability, high-precision printing capability, and good mechanical properties, demonstrating its great potential in DLP 3D printing. These properties make it an ideal bio-ink for 3D printing applications with complex structures and high precision requirements, especially in the fields of biomedical engineering and tissue engineering.

[0053] Example 3 Cross-linking mechanism of CMA-DHM and characterization of gel properties

[0054] Rheological analysis: The storage modulus (G') and loss modulus (G") of CMA and CMA-DHM gels at a strain of 0.2%-20% were determined by a rheometer in oscillation mode, with an angular frequency of 1 rad / s. At the same time, the dynamic viscosity of CMA and CMA-DHM gels was measured in the shear rate range of 0.1 to 100 / s, with an angular frequency of 1 rad / s and a strain of 1%.

[0055] Compression test: CMA and CMA-DHM gels were subjected to uniaxial compression tests using a universal testing machine. A hydrogel cylindrical sample with a size of 10 mm × 10 mm was loaded with a compressive force at a displacement rate of 20 mm / min until the sample strain reached 60%, and the stress-strain curve was recorded.

[0056] Swelling test: The swelling degree was calculated by measuring the weight of freeze-dried CMA and CMA-DHM gel after water absorption at different time points.

[0057] Anti-degradation test: CMA and CMA-DHM gel samples were incubated in 0.5 U / mL type I collagenase solution (0.05 M Tris-HCl buffer, pH 7.4, 37°C) for 10 days. Samples were taken out at different time points, freeze-dried and weighed to calculate the mass residual rate of the hydrogel and evaluate its degradation performance.

[0058] Infrared spectroscopy test: The freeze-dried CMA and CMA-DHM gel samples were mixed with potassium bromide and pressed into thin films, and their infrared spectra were measured using a Fourier transform infrared spectrometer (FTIR) to analyze the chemical structure of the samples.

[0059] The exact mechanism of DHM cross-linking collagen has not been fully reported, but considering that DHM contains highly reactive phenolic hydroxyl groups, its cross-linking mechanism may be similar to that of polyphenolic compounds. This mechanism includes non-covalent interactions such as hydrogen bonding and hydrophobic interactions, as well as the oxidation of phenolic hydroxyl groups to quinones, which then react with amino groups through Schiff base reactions and Michael addition reactions. Figure 3 As shown in a, the typical collagen characteristic peaks of CMA are shown. These peaks include the amide A band peak (3342 cm -1 ), represents CH and -NH 3 + Stretching vibration amide B band peak (3080.2cm -1 ), the amide I band peak (1660.4 cm) representing C=O stretching vibration and NH bending vibration -1 ), the amide II band peak (1561.5 cm) attributed to NH bending vibration and CN stretching vibration -1 ), and the amide III band (1237.6 cm) also represents the CN and NH vibrations. -1 ). In CMA-DHM gel, the amide A band peak shifted to 3344.4 cm -1 , the amide B band peak shifted to 3076 cm -1 , the amide I band peak shifted to 1663.7 cm -1 , which indicates that the amino and carboxyl groups in the CMA molecule underwent cross-linking reactions with DHM, including hydrogen bonding interactions.

[0060] After the reaction of DHM and CMA, the amide I band became broader and appeared at 1637 cm -1 This may be attributed to the C=N stretching vibration peak generated after the Schiff base reaction. In CMA-DHM gel, 1437.6 cm -1 The peak shifted and enhanced, which may be due to the covalent bond formed by DHM cross-linking, resulting in the enhancement of the bending vibration of CH or the stretching vibration of CN. In addition, CMA at 1129.6 cm -1 The peak of -1 This is due to the vibration of C-OH introduced by DHM and the enhanced vibration of CO caused by the new bonds generated by the reaction. In addition, a 1317 cm -1 The new absorption peak at 1025.5cm -1 and 955.5cm -1 Two strong absorption peaks appeared at DHM, which were attributed to the COC stretching vibration of DHM and were probably caused by the new covalent bonds generated by the reaction of DHM and CMA. These results indicate that the mechanism of DHM cross-linking CMA mainly includes non-covalent cross-linking including hydrogen bonding and hydrophobic interaction ( Figure 3 g) and DHM is oxidized to form quinone, which reacts with the amino group on collagen to undergo Schiff base reaction and Michael addition reaction ( Figure 3 h).

[0061] The properties of CMA and CMA-DHM gels were studied. The storage modulus and loss modulus of CMA-DHM gels were significantly higher than those of CMA gels without DHM crosslinking, indicating that CMA-DHM gels have higher viscoelasticity, and this effect is consistent under different strains ( Figure 3 b). The dynamic viscosity test showed that the dynamic viscosity of CMA-DHM gel at each shear rate was significantly higher than that of CMA gel, and decreased with the increase of shear rate, showing obvious shear thinning characteristics ( Figure 3 c). The stress-strain curves further confirmed that CMA-DHM gel had higher compressive stress than CMA at different strains and greater compressive fracture strength, indicating that the mechanical properties of CMA-DHM were significantly enhanced ( Figure 3 d). The swelling results showed that the swelling rate of CMA-DHM gel was significantly lower than that of CMA gel, suggesting that DHM cross-linking may increase the cross-linking density of the gel, thereby reducing the water absorption of the gel ( Figure 3 e). In the collagenase solution, the mass residual rate of CMA-DHM gel was significantly higher than that of CMA gel, indicating that CMA-DHM gel has stronger resistance to biological enzymatic hydrolysis ( Figure 3f). These results fully demonstrate that DHM cross-linking effectively improves the properties of CMA gel, including mechanical strength and anti-degradation properties.

[0062] Example 4 Biocompatibility of CMA-DHM ink

[0063] Preparation of cell-carrying ink: Soak the freeze-dried CMA in 75% ethanol for 30 minutes and air-dry at room temperature. Add DMEM containing 0.5M acetic acid and stir to dissolve for 24 hours, then adjust to neutral with high concentration NaOH to obtain a sterile CMA solution; add 25mg / mL dihydromyricetin to CMA to prepare CMA-DHM complex, so that the final concentration of DHM is 0.1mg / mL, and add photoinitiator LAP and FBS, and centrifuge to remove bubbles. Culture L929 cells to 80-90% confluence, digest the cells with 0.25% trypsin, and centrifuge to obtain a cell pellet. Resuspend the cells (1×10 7 cells / mL), and the cell-containing bio-ink is obtained. The cell-containing ink used for printing needs to be additionally added with ultraviolet absorber UA.

[0064] CCK 8 experiment: Pipette 100μL of cell-loaded bio-ink into a 96-well plate and add it vertically to avoid bubbles. Place the 96-well plate on a 405nm UV lamp for curing (30s) and prepare 6 replicate wells. Immediately after curing, wash with complete culture medium (DMEM, 5% FBS, 1% double antibody) three times. Add 200μL of complete culture for the last time and place in a 37℃ incubator for culture. Change the culture medium every day, and remove the well plate on the 1st, 4th, and 7th days, remove the culture medium, add 100μL of CCK 8 working solution diluted in DMEM, and culture at 37℃ for 1.5h in the dark; use an enzyme reader to detect the absorbance at 450nm.

[0065] Live and dead cell staining: Pipette 250 μL of cell-laden bio-ink and evenly add it to the confocal dish, and place it on a 405nm UV lamp for curing (30s). Immediately after curing, wash with complete culture medium (DMEM basal culture medium, 5% FBS, 1% double antibody) three times. Add 1mL of complete culture medium for the last time and place it in a 37°C incubator for culture. Change the culture medium every day, and remove it on the 1st, 4th, and 7th days, and remove the culture medium. First wash with 1×AB buffer for 5 minutes, add calcein (live cell staining) and PI (dead cell staining) according to the instructions, and stain at 37°C for 1 hour in the dark. Use a laser confocal microscope to capture images and count cells using Image J.

[0066] Cell-carrying printing: Add the cell-containing ink to the ink tank of the DLP printer, and use the default slicing parameters and the pre-adjusted printing parameters to print a 10×10mm grid. After printing, quickly place the grid in a culture medium, cure it under UV light for about 20 seconds, wash the grid three times, add fresh culture medium, and culture it in a 37°C cell culture incubator. Change the culture medium every day, and take it out on the 1st, 4th, and 7th day, remove the culture medium, and perform live and dead cell staining according to the instructions. Use a laser confocal microscope to take images, and use the ruler provided by the microscope software to measure the length and width of the grid on the 1st, 4th, and 7th day to observe the swelling of the grid. The grid printed on the first day was fused with the CAD model (10×10mm) in the PS software to observe the printing accuracy. At the same time, the grid printed on the first day was photographed with a bright field image by laser confocal, adjusted to black and white in Image J software, and a green straight line was drawn. The grayscale of the straight line was calculated by Image J software to observe the printing quality.

[0067] The results are as follows Figure 4 As shown, the cells encapsulated by CMA and CMA-DHM inks were green on days 1, 4, and 7, with almost no red, indicating that the cells were in a viable state ( Figure 4 a). Further quantitative analysis revealed that the cells in CMA-DHM proliferated significantly compared to those in CMA ( Figure 4 b). This result is consistent with the results of the CCK-8 experiment ( Figure 4 c). Live and dead cell staining results of cell-loaded printed grids show that the cell survival rate was high and evenly distributed in the grid on the first day after printing, which was well integrated with the CAD model ( Figure 4 d). On the 4th and 7th days of culture after printing, the cells within the grid proliferated significantly ( Figure 4 e). Further analysis of the printing accuracy of the cell-carrying printing revealed that the edges of each mesh of the printed grid were clear ( Figure 4 f), the grid side length is uniform, the printing quality is stable, and there is no statistical difference in the length and width of each side ( Figure 4 g), proving that the addition of cells has no effect on printing accuracy. The grid length after printing is widened, which is mainly caused by gel swelling and may also be affected by cell growth ( Figure 4 h). There was no significant difference in the side length between the 4th day and the 7th day (p>0.05), indicating that the printed gel almost reached swelling equilibrium on the 4th day. Overall, these experimental results show the feasibility of CMA-DHM as a bio-ink for cell printing. CMA-DHM has higher biocompatibility, can promote cell proliferation, and has higher printing accuracy and stability, showing its potential as a tissue engineering bio-ink.

[0068] Example 5 Antioxidant capacity of CMA-DHM gel

[0069] Sample preparation: Human dermal fibroblasts (HDFs) were cultured to 80-90% confluence, digested with 0.25% trypsin and centrifuged to obtain a cell pellet. The cells were resuspended in culture medium to prepare a cell suspension of 1×10^6 cells / mL. CMA and CMA-DHM gels were prepared and washed three times with PBS, and then soaked in DMEM for 24 hours. 100 μL of cell suspension was evenly spread on the surface of the gel, and fresh culture medium was added after overnight culture, and culture was continued for 3 days.

[0070] Antioxidant-related experiments: The culture medium in the samples was removed, and the CMA and CMA-DHM samples were treated with H2O2 (88 μM) solution diluted in DMEM for 40 minutes, and the untreated CMA was used as a blank control (Control). Subsequently, the DCFH-DA (10 μM) fluorescent probe was added, and the Control, CMA, and CMA-DHM were stained in the dark for 60 minutes. After staining, the staining solution was removed, the samples were washed with PBS, and fluorescence images were taken using a laser confocal microscope. The fluorescence of different samples was semi-quantitatively analyzed using Image J. At the same time, the samples cultured in 96-well plates were stained according to the same operation and then quantitatively analyzed using an ELISA reader. In addition, live and dead cell staining was performed on the three groups of samples of Control, CMA, and CMA-DHM. The culture medium in the samples was removed, the samples were washed with 1×AB buffer, calcein and PI dyes were added according to the instructions, and the samples were treated in the dark for 1 hour. Live and dead cell images were taken using a laser confocal microscope, and dead cells were quantitatively analyzed using Image J.

[0071] Cytoskeleton staining: Remove the medium from CMA and CMA-DHM samples and wash with PBS. Stain the cells on the gel for actin using rhodamine-labeled phalloidin staining reagent, and counterstain the nuclei with Hoechst 33342 staining solution. Obtain fluorescence images using a laser confocal microscope, and count cells using Image J.

[0072] like Figure 5 As shown, H 2 O 2 Treatment resulted in the generation of reactive oxygen species (ROS). However, the sustained release of DHM, an effective ROS scavenger, by CMA-DHM gel created an oxidative stress-free environment that was beneficial for cell proliferation in 3D culture ( Figure 5 a). Therefore, after 3 days of culture, the number of cells grown on CMA-DHM gels was almost twice that on CMA gels ( Figure 5b). Cytoskeleton staining further demonstrated the effect of CMA-DHM gel in promoting cell adhesion, proliferation and spreading ( Figure 5 c).

[0073] The superior antioxidant capacity of CMA-DHM gel was characterized by DCFH-DA and live-dead cell staining. Figure 5 d) The results show that after the same H 2 O 2 After treatment, the CMA group showed strong fluorescence, indicating that the cells were in a state of high reactive oxygen, while the fluorescence intensity of the CMA-DHM group was not only lower than that of the CMA group, but even lower than that of the untreated control group. Figure 5 e) and fluorescence intensity quantification ( Figure 5 f) This was further proved by live and dead cell staining of the three groups of samples, and the results showed that there were a large number of dead cells in the CMA group, which was related to the high oxidative stress level of the cells ( Figure 5 d). Further semi-quantitative analysis found that the proportion of dead cells in the CMA group was significantly higher than that in other groups, indicating that reducing oxidative stress is important for cell survival ( Figure 5 g). These results demonstrate the potential of DHM as a multifunctional crosslinker, which not only improves the mechanical properties of the gel but also endows the gel with excellent antioxidant capacity, thus further enhancing the biocompatibility and bioactivity of the CMA system.

[0074] Example 6 Diabetes Wound Healing Study

[0075] A double-layer artificial skin model with a diameter-to-height ratio of 9:2.5 was designed using 3D modeling software ( Figure 6 a). Prepare bio-ink and add HDFs cells, and prepare samples by DLP 3D printing for animal experiments and in vitro culture studies. After 14 days of in vitro culture, the printed gel maintained good structural integrity and high resolution. Live and dead cell staining showed that 14 days of culture resulted in a large number of cell proliferation in the gel, and almost no dead cells ( Figure 6 a), indicating that the system has potential for clinical application.

[0076] A drug-induced diabetes model was established using 150-200 g SD male rats by a single intraperitoneal injection of 65 mg / kg STZ. Adequate drinking water and feed were provided daily, and the bedding was changed regularly. After 3 weeks, a full-thickness skin defect wound model with a diameter of 9 mm was established. The control group did not receive any scaffold treatment, and the printed CMA, CMA-DHM, and CMA-DHM@HDFs were implanted into the wound. A wound model was established in healthy rats without diabetes as a control. All groups used 3M transparent dressings to fix the wound, and the dressings fell off on their own during the subsequent feeding period without replenishment. The control group had obvious wound infection within 7-10 days. To avoid death due to severe infection, all groups of rats were systemically disinfected and the wounds were treated with iodine cotton balls.

[0077] The wounds were photographed on days 0, 4, 7, 10, 15, and 18 and organized into 2 cm × 2 cm images to observe the wound healing over time. ImageJ was used to analyze the wound healing process and wound closure rate. Figure 6 As shown in Figure b, compared with the acute wound control group, the diabetic control group showed significant delayed wound healing on days 4, 7, 10, 15, and 18, which is consistent with the characteristics of diabetic wounds that are difficult to heal. The wound closure speed of the three groups treated with stents at different time points was faster than that of the control group, showing the effectiveness of stent treatment. Compared with the CMA group, the wound closure rate of the CMA-DHM group was significantly accelerated due to its excellent antioxidant capacity ( Figure 6 c), especially during the inflammatory phase between days 4 and 7 ( Figure 6 d). In addition, the cell-loaded CMA-DHM@HDFs group exhibited faster healing speed and better healing quality during the wound healing process, highlighting the potential advantages of cell-loaded artificial skin in the treatment of diabetic wounds.

[0078] In summary, the present invention provides a multifunctional collagen bio-ink for DLP 3D printing, wherein the bio-ink comprises the following components: 0.5%-1.5% m / v methacryloyl collagen solution, 0.125%-0.5% m / v photoinitiator, 0.025%-0.1% m / v UV absorber and 0.05-0.4 mg / mL dihydromyricetin solution, wherein the bio-ink has good fluidity, higher mechanical properties, lower swelling ratio, and better anti-degradation performance; it can print complex three-dimensional models, maintain micron-level printing accuracy while having higher cell-carrying biocompatibility and structural stability, can promote cell proliferation in printed gel, and demonstrates its potential as a tissue engineering bio-ink; the bio-ink printing of the present invention The post-gel scaffold has excellent antioxidant properties, can effectively remove reactive oxygen species (ROS), provide a 3D culture environment without oxidative stress for human fibroblasts (HDFs), significantly promote the proliferation and spreading of HDFs on the gel, and further improve the biological activity of the collagen gel; the artificial skin prepared by the bio-ink loaded with human fibroblasts (HDFs) described in the present invention, after 14 days of culture, the gel still maintains good structural integrity, and the cells in the gel proliferate significantly, and the cell survival rate is extremely high; it is applied to the treatment of diabetic wounds, showing faster healing speed and better healing quality, highlighting the potential advantages of cell-loaded artificial skin in the treatment of diabetic wounds, and has good clinical application potential.

Claims

1. A multifunctional collagen bio-ink for DLP 3D printing, characterized in that: The biological ink comprises the following components: 0.5%-1.5% m / v methacryloyl collagen solution, 0.125%-0.5% m / v photoinitiator, 0.025%-0.1% m / v ultraviolet absorber and 0.05-0.4 mg / mL dihydromyricetin solution.

2. The bio-ink according to claim 1, characterized in that: The collagen is bovine collagen.

3. The bio-ink according to claim 2, characterized in that: The bovine collagen is type I bovine collagen, and / or type II bovine collagen, and / or type II bovine collagen.

4. The bio-ink according to claim 1, characterized in that: The photoinitiator includes one or more of LAP, TPO and I2959.

5. The bio-ink according to claim 1, characterized in that: The ultraviolet absorber includes lemon yellow and / or food coloring brilliant blue.

6. The method for preparing the biological ink according to any one of claims 1 to 5, characterized in that: The steps include: (1) Dissolve the freeze-dried collagen sponge in acetic acid solution and stir at low temperature until it is completely dissolved; (2) adding methacrylic anhydride dropwise to the collagen solution obtained in step (1), adjusting the pH to 4-9, reacting at low temperature for 24-48 hours, dialyzing, and freeze-drying to obtain methacrylylated collagen; (3) The methacryloyl collagen obtained in step (2) is dissolved in acetic acid solution to a concentration of 0.5%-1.5% m / v, and sodium hydroxide solution is added to adjust the pH of the solution to neutral; 0.125%-0.5% m / v photoinitiator, 0.025%-0.1% m / v ultraviolet absorber and 0.05-0.4 mg / mL dihydromyricetin solution are added, mixed evenly, and centrifuged to remove bubbles to obtain CMA-DHM bio-ink.

7. Use of the bio-ink as described in any one of claims 1 to 5 in the preparation of one or more of tissue engineering materials, hemostatic materials, wound dressings, and drug delivery materials.

8. The use according to claim 7, characterized in that The tissue engineering material includes one or more of artificial skin, artificial blood vessels, cartilage and bone repair materials and corneal transplantation materials.

9. An artificial skin for treating chronic diabetic wounds, characterized in that: The artificial skin is obtained by adding human fibroblasts to the biological ink described in any one of claims 1 to 5 and printing it through DLP 3D printing.

10. Use of the artificial skin as claimed in claim 9 in treating chronic diabetic wounds.

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