DLP 3D printing bio-ink of Zn < 2 + > coordinated collagen, preparation method and application

Abstract

The invention belongs to the technical field of biology, and relates to DLP 3D printing bio-ink of Zn < 2 + > coordinated collagen, a preparation method and application, the bio-ink comprises the following components: 0.1-1% m / v of a methacrylic acid collagen solution, 0.1-0.5% m / v of a photoinitiator, 0.05-0.5% m / v of an ultraviolet absorbent and 0.01-0.1% m / v of a Zn < 2 + > solution, the hydrogel printed by the bio-ink has the advantages of high mechanical property, high degradation resistance, good biocompatibility, mildness and no irritation to skin and the like, the preparation conditions are simple and mild, and the biological activity of collagen is reserved; according to the bio-ink, a skin care product is customized in a personalized mode through the DLP 3D printing technology, the application time is prolonged, the bio-ink has more lasting moisturizing and anti-aging capacity, the active ingredient acetyl hexapeptide-8 is added, dynamic wrinkles, static wrinkles and fine wrinkles can be improved, synthesis of collagen, elastin and hyaluronic acid is promoted, and the effect of tightening the skin is achieved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a Zn 2+ DLP 3D printing bio-ink with coordinated collagen, preparation method and application. Technical Background

[0002] With the growing demand for personalization and customization, additive manufacturing technology, especially 3D printing technology, has been widely used in many fields such as medicine, food and manufacturing around the world. The cosmetics industry has also begun to adopt this technology to develop products that meet consumers' personalized skin care needs. At present, many companies including Neutrogena, AmorePacific and Procter & Gamble (P&G) have used 3D printing technology to customize skin care products according to users' facial contours and specific skin care needs. Researchers such as Erieta Manousi and Junli Chen have developed extrusion-based 3D printed hydrogel facial patches. [1] and multifunctional mask [2] In addition, fused deposition modeling (FDM) and stereolithography (SLA) 3D printing technologies have been used to make personalized salicylic acid patches. [3] Although digital light processing (DLP) 3D printing technology has significant advantages due to its superior printing speed, printing accuracy and printing resolution, its application in skin care products is still in its preliminary stage.

[0003] The preparation of bio-ink is the most critical step in DLP 3D bioprinting, and the bio-ink that can be used for DLP 3D bioprinting of medical materials has strict requirements and should have the following characteristics: (1) Printability: Bio-ink can accurately control the performance of additive molding in spatial and temporal dimensions, and quickly solidify and mold; (2) The printed biomedical materials should have good biocompatibility; (3) The mechanical properties of the printed biomedical materials are stable; (4) The printed biomedical materials have degradable properties; (5) The printed biomedical materials have good biomimetic functions. At present, bio-inks used in 3D printing technology include bio-inks prepared from gelatin, such as the invention patent CN202310232743.0 disclosed with methacryloyl gelatin, polyethylene glycol diacrylate, cellulase, Lactobacillus pentosus and liquid culture medium. However, since gelatin is a denatured collagen, it loses the biological activity of collagen, which limits its application. Other synthetic bio-inks made of thermoplastic degradable polyester plastics lack biocompatibility and biomimetic functions, cannot provide a suitable environment for the growth of active cells, and are difficult to use to prepare medical tissues / organs with biocompatibility and biomimetic functions. Therefore, it is crucial to develop a bio-ink with high bioactivity.

[0004] Considering that collagen is a component with multiple biologically active effects on the skin, it can promote cell proliferation, adhesion, migration and differentiation, and has unique hydrophilicity and film-forming properties, it is particularly important to develop a methacrylic anhydride-modified collagen (CMA) bio-ink. This new bio-ink can achieve effective intermolecular cross-linking under ultraviolet irradiation, and therefore shows broad application prospects in DLP 3D printing. However, how to solve the low solubility and insufficient mechanical strength of collagen to achieve its efficient application in DLP 3D printing is still an important technical challenge.

[0005] References:

[0006] [1]Manous,et al.Development and in vivo evaluation of 3D printedhydrogel patches for personalized cosmetic use based on skin type.Journal ofDrug Delivery Science and Technology.2024,92:105306

[0007] [2]Chen et al.Preparation and efficacy verification of threedimensional printed partitioned multi-effect precision-care facial gelmask.International Journal ofCosmetic Science,2024,46(2):209-227

[0008] [3]Goyanes et al. 3D scanning and 3D printing as innovative technologies for fabricating personalized topical drug delivery systems. Journal of controlled release, 2016, 234: 41-48 Summary of the invention

[0009] In view of the above problems, the primary purpose of the present invention is to provide a Zn 2+DLP 3D printing bio-ink with collagen, the bio-ink comprising the following components: 0.1-1% m / v methacrylated collagen solution, 0.1-0.5% m / v photoinitiator, 0.05-0.5% m / v UV absorber and 0.01-0.1% m / v Zn 2+ Solution.

[0010] Preferably, the bio-ink comprises the following components: 0.5-1% m / v methacrylated collagen solution, 0.25-0.5% m / v photoinitiator, 0.1-0.5% m / v UV absorber and 0.01% m / v Zn 2+ Solution.

[0011] Preferably, the collagen is yak collagen.

[0012] Preferably, the yak collagen is type I yak collagen, and / or type II yak collagen, and / or type II yak collagen.

[0013] Preferably, the photoinitiator includes one or more of LAP, TPO, and I2959.

[0014] Preferably, the photoinitiator is LAP.

[0015] Preferably, the ultraviolet absorber includes one or more of UA, lemon yellow, and food coloring brilliant blue.

[0016] Preferably, the ultraviolet absorber is UA.

[0017] The second object of the present invention is to provide a method for preparing the bio-ink, comprising the following steps:

[0018] (1) dissolving the freeze-dried collagen sponge in an acetic acid solution to prepare a collagen solution, and slowly stirring to dissolve;

[0019] (2) adjusting the pH of the collagen solution obtained in step (1) to 7-9, adding methacrylic anhydride dropwise, and maintaining the pH of the solution at 7-8; reacting in an ice bath for 24 hours, dialyzing, and freeze-drying to obtain methacrylated collagen;

[0020] (3) Prepare 0.1-1% m / v methacrylated collagen solution with 0.5M acetic acid solution, add 6M sodium hydroxide solution to adjust the pH of the solution to neutral, add 0.1-0.5% m / v photoinitiator, 0-0.5% m / v UV absorber and 0.01-0.1% m / v Zn 2+ The solution was mixed evenly and centrifuged to remove bubbles to obtain CMA-Zn 2+ Bio-ink.

[0021] Preferably, the pH of the collagen solution in steps (2) and (3) is adjusted using sodium hydroxide solution.

[0022] The third object of the present invention is to provide a skin repair material, which is obtained by adding active ingredients to the biological ink described in any one of claims 1 to 5 and printing it through DLP 3D.

[0023] The fourth object of the present invention is to provide the application of the biological ink in the preparation of skin repair materials, bone-cartilage repair materials, and tissue engineering.

[0024] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a Zn 2+ DLP 3D printing bio-ink with collagen, the bio-ink comprising the following components: 0.1-1% m / v methacrylated collagen solution, 0.1-0.5% m / v photoinitiator, 0.05-0.5% m / v UV absorber and 0.01-0.1% m / v Zn 2+ Solution. The bio-ink is prepared by combining metal ions and chemical cross-linking to obtain collagen hydrogel, which has the advantages of high mechanical properties, strong anti-degradation ability, good biocompatibility, mild and non-irritating to the skin, and the preparation conditions are simple and mild, which retains the complete triple helix structure and biological activity of collagen to the greatest extent; the bio-ink described in the present invention uses DLP 3D printing technology to customize skin care products, overcoming the defect that popular skin care products cannot meet individual needs; compared with ordinary skin care products using base cloth as a carrier, collagen is used as a hydrogel carrier to extend the application time and has a more lasting moisturizing and anti-aging ability; in addition, after adding the active ingredient acetyl hexapeptide-8, it can also improve dynamic lines, static lines and fine lines, promote the synthesis of collagen, elastin and hyaluronic acid, and has the effect of tightening the skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The elements and concentrations of the metal ion coordination cross-linking described in the present invention are screened.

[0026] Note: (A) respectively add (a) Cu 2+ (b) Al 3+ 、(c)Zn 2+ CMA bio-inks and photocured images of the elements. (B) Adding different concentrations of Zn 2+ The CMA bio-ink and photocuring images after treatment. (a) low concentration, (b) high concentration.

[0027] Figure 2 The CMA and CMA / Zn of the present invention 2+ Characterization of bioinks and their hydrogels.

[0028] Note: (A) Bio-ink CMA / Zn 2+ Printability and (B) rheological properties, (C) CMA / Zn 2+ Stress-strain curves, (D) degradation resistance, (E) swelling properties, and (F) element distribution diagram of the hydrogel.

[0029] Figure 3 The CMA / Zn 2+ Printability of bio-inks.

[0030] Note: (A) 3D model and printed image of a gecko. (B) 3D model of a pair of nuts and bolts and its printed and matched images. (C) 3D model of a mesh bracket with small holes and its printed image.

[0031] Figure 4 The CMA / Zn 2+ Cell encapsulation and printing of bio-inks.

[0032] Note: (A) CMA and CMA / Zn 2+ Live / dead cell staining at 0, 4, and 7 days after bio-ink cell encapsulation and (B) quantification of cell density using Image J software. (C) CMA and CMA / Zn 2+ Absorbance at 450 nm at 0, 4, and 7 days after bio-ink cell encapsulation. (D) Human dermal fibroblasts seeded on CMA and CMA / Zn 2+ Cytoskeleton staining of hydrogel after 3 days. (E) CMA / Zn 2+ Live / dead cell staining after bio-ink-loaded cell printing;

[0033] Figure 5 The personalized skin care solution represented by the eye mask described in the present invention.

[0034] Note: (A) 5 steps to develop personalized skin care products. (B) Analysis of periocular skin wrinkles using the VISIA facial scanner. (C) Human face model built using Blender software. (D) Design of a 3D eye mask model based on the face model. (E) Personalized bio-ink with anti-wrinkle properties is achieved by adding AHP-8 (0.05%). (F) DLP-based printing process. (G) Printed personalized eye mask, which can fit closely to the skin (G1) and bend along the skin contour (G2). (H) Personalized eye mask used on volunteers and (F, J) wrinkle images captured by the VISIA facial scanner on days 7 and 14;

[0035] Figure 6 In vivo validation of the printed eye mask of the present invention on an anti-aging mouse model.

[0036] Note: (A) Establishment of aging mouse model and treatment plan. (B) Monitoring of mouse weight within 4 weeks. (C) Control group, model group, CMA / Zn 2+ Group, CMA / Zn 2+ Skin texture of mice in the CMA / Zn / AHP-8 group. (D) 2+ Group, CMA / Zn 2+ High-frequency ultrasound images of the skin of mice in the / AHP-8 group, (E) dermal density, (F) transepidermal water loss, and (G) hydration;

[0037] Figure 7 Histological analysis of the skin of the mouse anti-aging model described in the present invention.

[0038] Note: (A) Control group, model group, CMA / Zn 2+ Group, CMA / Zn 2+ Representative images of H&E staining and Masson staining in the / AHP-8 group. (B) Control group, model group, CMA / Zn 2+ Group, CMA / Zn 2+ Collagen deposition, (C) HYP content, (D) SOD level, and (E) MDA content in the skin of mice in the / AHP-8 group. Specific implementation methods

[0040] The technical solution of the present invention is further described below in conjunction with the implementation modes, but the protection scope of the present invention is not limited to the following.

[0041] In the following examples, unless otherwise specified, the methods used are all conventional methods;

[0042] In the following examples, unless otherwise specified, the reagents used are conventional reagents and can be purchased from the market.

[0043] 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 yak collagen, and collagen obtained by any other means may be used, including type I collagen, type II collagen, type III collagen, etc.

[0044] 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.

[0045] Example 1: Elements and concentration screening of collagen DLP 3D printing bio-ink

[0046] 200 mg of freeze-dried collagen was dissolved in 100 mL of 0.5 M acetic acid, and the solution was stirred at 4 ° C until completely dissolved. Subsequently, 1 mL of methacrylic anhydride (MA) was added to the collagen solution for 24 hours, maintaining the reaction temperature at 4 ° C and the reaction pH at 8-9. The solution after the reaction was dialyzed to remove residual MA. The resulting CMA solution was lyophilized and stored at 4 ° C for further use.

[0047] CMA powder was dissolved in 0.5 M acetic acid, stirred at 4 °C for 24 h, and then neutralized with NaOH to obtain CMA solution. 2+ 、Al 3+ and Zn 2+ Slowly add 0.5% CMA solution, then add 0.25% photoinitiator (LAP) and 0.05% UV absorber (UA) to obtain the corresponding bio-ink. The above bio-inks are placed under a 405nm wavelength UV lamp for curing for 30s to prepare a hydrogel with an inherent container shape.

[0048] The experimental results show that Cu 2+ ( Figure 1 Aa) and Al 3+ ( Figure 1 Ab) After adding CMA solution, collagen quickly formed disordered and rough flocculent precipitates, and could not form gel after UV light; while Zn 2+ ( Figure 1 Ac) After adding CMA solution, the collagen solution has a uniform texture and can form a gel after ultraviolet light.

[0049] In order to explore Zn 2+ The effect of concentration on CMA bio-ink. We used low concentration (0.01%) and high concentration (0.1%) of Zn 2+ The solutions were added to the CMA solution, and the bio-ink and hydrogel were prepared according to the above method; the experimental results showed that low and high concentrations of Zn 2+ The bio-ink prepared by the solution has a uniform texture and no protein precipitation ( Figure 1 Ba), but high concentrations of CMA / Zn 2+ The solution cannot be UV cured ( Figure 1 Bb), and select the low concentration for subsequent experiments.

[0050] Example 2: Preparation of collagen DLP 3D printing bio-ink

[0051] Collagen DLP 3D printing bio-ink is mainly composed of CMA solution, photoinitiator LAP, UV absorber UA and Zn 2+In order to explore the effects of CMA solution concentration, photoinitiator LAP concentration and UV absorber UA concentration on 3D printing, we 2+ When the solution concentration was determined to be 0.01%, CMA / Zn was prepared according to Table 1. 2+ Bio-ink.

[0052] Among them, bio-inks 1, 2, and 3 cannot form hydrogels with specific shapes due to their low protein concentration and poor mechanical properties; bio-inks 4, 5, and 6 have moderate fluidity and high printing precision, but bio-ink 5 takes a long time to print, bio-ink 6 has low printing precision, and high concentrations of LAP have certain cytotoxicity; bio-inks 7, 8, and 9 have too high viscosity and low printing precision. Therefore, we chose bio-ink 4 as the printing ink for subsequent experiments.

[0053] Table 1 Content of each component in different bio-inks

[0054] Bio-ink serial number CMA Concentration Photoinitiator LAP concentration Light absorber UA concentration Bio-ink 1 0.1％ 0.5％ 0.05％ Bio-ink 2 0.1％ 0.25％ 0.05％ Bio-ink 3 0.1％ 0.5％ 0.5％ Bio-ink 4 0.5％ 0.25％ 0.1％ Bio-ink 5 0.5％ 0.25％ 0.5％ Bio-ink 6 0.5％ 0.5％ 0.05％ Bio-ink7 1％ 0.25％ 0.1％ Bio-ink8 1％ 0.1％ 0.5％ Bio-ink9 1％ 0.5％ 0.1％

[0055] CMA / Zn using DLP 3D printer 2+ Accurate printing of ink. Use CAD software to design 3D models and save them as STL files. Then slice these files in the Z direction and project them through a projector to achieve layer-by-layer printing of the 3D design model. The printing conditions are set to slice thickness of 100-200μm and UV intensity of 15-25mW / cm 2 The layer curing time is 10-20s; to ensure thorough cross-linking, the hydrogel after printing will be cured by UV light for 10-20s.

[0056] Example 3. Characterization of collagen DLP 3D printing bio-ink and its hydrogel properties

[0057] 1. Experimental Procedure

[0058] Rheological tests were performed on a parallel plate rheometer. -1 Time sweep tests were performed at frequency and 1% strain to monitor CMA and CMA / Zn 2+ Storage modulus (G') and loss modulus (G") of the bioink under UV irradiation (405 nm).

[0059] Compressive stress-strain measurements were performed using a tensile machine at room temperature. 2+ The hydrogel was printed into a 2.5 mm × 13 mm cylindrical shape. A compressive strain rate of 10 mm / min was applied during the experiment.

[0060] The freeze-dried hydrogels were measured before immersion in deionized water (m0) and at different time points during immersion (m t ) to detect the swelling rate of the hydrogel. The swelling capacity is calculated by the following formula:

[0061]

[0062] The hydrogel was immersed in type I collagenase solution (0.5 U / mL, 0.05 M Tris-HCl buffer, pH = 7.4), incubated at 37°C for 9 days, and freeze-dried and weighed to study its degradation behavior. The degradation rate was calculated as follows:

[0063]

[0064] Where m0 represents the initial weight of the sample, m t Indicates the weight at each time point.

[0065] Scanning electron microscopy observation of CMA / Zn 2+ The microstructure and element distribution of C, N, O, and Zn. 2+ The hydrogel sample was cut and stuck on the conductive glue, sprayed with gold for 60 s, and then photographed at 2 kV and 140×.

[0066] 2. Experimental results

[0067] CMA is considered to be the preferred material for 3D bioprinting due to its excellent bioactivity. However, low concentrations of CMA often lead to insufficient mechanical strength of DLP 3D printing. 2+ Combined with the development of a new type of CMA / Zn 2+ Bio-ink. This bio-ink was then used in a DLP-based 3D printer and successfully printed a 3D model with complex ridges ( Figure 2 A). We performed time sweep rheology tests to evaluate the CMA and CMA / Zn 2+ In situ gelation process of bio-ink under UV irradiation ( Figure 2 B). After UV irradiation, CMA and CMA / Zn 2+ The storage modulus (G') and loss modulus (G″) of Zn increased rapidly and stabilized at the maximum value within 10 s. It is worth noting that 2+ The addition of trace amounts of can significantly improve the mechanical strength of CMA.

[0068] The compressive stress-strain curve is Figure 2 C shows that CMA / Zn 2+The compressive strength at break of the hydrogel (26.2 kPa) is greater than that of the CMA hydrogel (22.9 kPa), indicating that Zn 2+ The addition of α-glucose significantly enhanced its compressive strength. This demonstrates the successful development of a new collagen-based bioink with sufficient mechanical durability for DLP bioprinting.

[0069] CMA and CMA / Zn 2+ The enzymatic degradation rate of the hydrogel is Figure 2 As shown in D, after incubation in type I collagenase solution for 9 days, CMA / Zn 2+ The degradation rate of the hydrogels was always lower than that of the CMA hydrogels. This observation suggests that the CMA / Zn 2+ The anti-biodegradation properties of the hydrogel were improved.

[0070] The swelling capacity of the hydrogel is Figure 2 E. CMA / Zn 2+ The equilibrium swelling ratio of the hydrogel (97.69±10.12) was significantly lower than that of the CMA hydrogel (141.87±8.80), indicating that the CMA / Zn 2+ The hydrogel has higher anti-swelling ability in water environment. CMA and CMA / Zn 2+ The hydrogels showed porous microstructures in SEM images, and the mapping images showed that CMA / Zn 2+ The composite material contains C, N, O and Zn elements, and Zn is evenly distributed throughout the collagen network ( Figure 2 F).

[0071] Example 4. Printability of collagen DLP 3D printing bio-ink

[0072] In order to show that CMA / Zn 2+ Taking advantage of the versatility and precision of bio-ink, we used DLP 3D printers to prepare three complex and challenging 3D models. Among them, a 2 cm long gecko model was carefully designed and printed ( Figure 3 A). The five fingers of the printed gecko are clear and distinct, indicating that CMA / Zn 2+ Bio-ink has a high printing resolution. In addition, a pair of bolts and nuts were printed together, and the two could be perfectly aligned after printing, highlighting the precision and accuracy of DLP 3D printing ( Figure 3 B). In addition, a mesh scaffold with 100 small holes was printed, and each hole of the scaffold was clearly visible ( Figure 3 C). All printed 3D structures corresponded precisely to the designed models in terms of pattern and size, demonstrating the novel CMA / Zn 2+ The potential of bioinks in 3D bioprinting applications.

[0073] Example 5. Biocompatibility of collagen DLP 3D printing bio-ink

[0074] 1. Experimental Procedure

[0075] Live / dead cell staining was used to detect cell viability. 6 L929 cells / mL were encapsulated in CMA and CMA / Zn 2+ In the bio-ink, CMA and CMA / Zn were obtained by UV irradiation for 30s. 2+ The hydrogels were washed three times with complete medium and incubated in a 5% CO2 incubator at 37°C, with the medium changed daily. Cell viability was detected by live / dead cell staining on days 0, 4, and 7, and images were taken using a laser confocal scanning microscope. Fluorescence images were used to quantify cell viability using Image J software.

[0076] CCK-8 assay was used to detect cell proliferation. 6 L929 cells / mL and CMA or CMA / Zn 2+ The bio-ink was mixed and added to a 96-well plate. UV light was used to cure the plate for 30 seconds, followed by washing with culture medium and incubation in an incubator at 37°C and 5% CO2. On days 0, 4, and 7, 100 μL of CCK-8 working solution was added to each well, and the absorbance was measured at 450 nm using a microplate reader.

[0077] Cytoskeleton staining was used to detect cell adhesion. 2+ The cells were placed on the hydrogel and co-cultured in an incubator at 37°C and 5% CO2. After 3 days of incubation, the cytoskeleton fibers were stained with rhodamine / phalloidin and the cell nuclei were stained with Hoechst 33342. Laser confocal microscopy was used to capture cell images.

[0078] 2. Experimental results

[0079] To evaluate the biocompatibility of the bio-inks, fibroblasts were encapsulated in CMA and CMA / Zn 2+ Live / dead cell staining in hydrogels ( Figure 4 A) CMA and CMA / Zn 2+ The L929 cells encapsulated in the hydrogels remained viable under 0.25% LAP and 30 s UV irradiation, indicating that the bio-inks were non-toxic to the cells. 2+Dead cells (red) were rarely observed in the hydrogels, indicating that these bio-inks have good biocompatibility. Quantitative analysis of live cells by live / dead cell staining images helps to understand the biocompatibility of CMA and CMA / Zn 2+ Proliferation trend of fibroblasts encapsulated in hydrogels. Quantitative analysis results showed that CMA and CMA / Zn 2+ Hydrogels have the effect of promoting cell proliferation ( Figure 4 B), this trend is consistent with the CCK-8 test results ( Figure 4 C). Cytoskeleton staining of human dermal fibroblasts (HDFs) was performed to further investigate their biocompatibility ( Figure 4 D) HDFs were inoculated with CMA and CMA / Zn 2+ On the surface of the hydrogel, the f-actin fibers were highly spread and elongated after 3 days of culture, indicating that the hydrogel can promote the adhesion of HDFs cells and has the potential to serve as a three-dimensional scaffold for cell growth. These results all indicate that CMA / Zn 2+ Bio-ink has good biocompatibility and bioactivity.

[0080] In addition, we used biocompatible CMA / Zn 2+ Bio-ink was used for bioprinting of cells. We designed a mesh scaffold with micron-sized pores and then inoculated cells with CMA / Zn 2+ The bio-inks were mixed and then the cell-containing bio-inks were DLP bioprinted using pre-adjusted parameters. The fibroblast-loaded mesh scaffolds that were quickly and accurately printed showed extremely high structural integrity ( Figure 4 E). Images taken with a confocal laser scanning microscope (CLSM) show that cells are evenly distributed within the scaffold and that cells remain viable 24 hours after printing. The enlarged image shows that there is no obvious damage to the cells, further emphasizing the CMA / Zn for DLP printing. 2+ Biocompatibility of bio-ink. Precise printability and excellent biocompatibility indicate that CMA / Zn 2+ Bio-ink is expected to be used to manufacture cell-loaded tissue structures through DLP 3D printing technology.

[0081] Example 6: Collagen DLP 3D Printing Bio-ink for Personalized Treatment

[0082] 1. Experimental Procedure

[0083] Personalized eye masks were developed through personalized skin care experiments. Initially, the volunteers' faces were scanned with a digital camera and the relevant data was obtained. Then, an accurate 3D facial model was processed and constructed using Blender 3.5 software. Based on this model, a personalized eye mask 3D model that conforms to the facial contours of the volunteers was carefully designed. 2+ The ink was supplemented with the famous anti-wrinkle peptide: acetyl hexapeptide-8 (AHP-8) to print a personalized eye mask that matches the volunteer's facial features. 2+ On days 0, 7, and 14 after application of the AHP-8 eye mask, facial images were captured using a VISIA facial scanner to record changes in skin condition.

[0084] 2. Experimental results

[0085] We demonstrated the use of CMA / Zn 2+ Bio-inks use DLP 3D printing technology to develop personalized skin care solutions ( Figure 5 A). The process involves several steps: face scanning (S1), 3D modeling (S2), skin care formula design (S3), fast DLP printing (S4), and personalized skin care application (S5). Customizing personalized skin care products for different facial contours can ensure the best contact between skin care ingredients and the skin, enhancing the absorption and efficacy of drugs. In addition, compared with traditional mass production, 3D printing of personalized cosmetics provides more precise material use, reduces waste, and achieves a sustainable production process.

[0086] To verify the feasibility of a personalized skin care program, we created a personalized eye mask. The VISIA facial scanner showed that the initial score of wrinkles around the eyes was 17.944 ( Figure 5 B). Subsequently, the volunteer's facial model was generated using Blender 3.5 software ( Figure 5 C), and based on the facial model, a 3D eye mask model was customized to fit the volunteer's facial curve, ensuring the most appropriate eye mask size and shape ( Figure 5 D) To solve the wrinkle problem, we sterilized CMA / Zn 2+ Anti-wrinkle peptide (acetyl hexapeptide-8AHP-8) is added to the ink formula. This peptide can disrupt muscle contraction and stimulate collagen synthesis, thereby reducing wrinkles ( Figure 5 E). Then, a DLP printer is used to make a personalized eye mask ( Figure 5 F). The images show that the printed eye mask can fit the skin precisely ( Figure 5 G1) and has high adhesion properties ( Figure 5 G2). After being applied to the volunteer's eyes, the eye mask adhered firmly to the skin around the eye socket ( Figure 5 H). Notably, the volunteers experienced a reduction in wrinkle scores around their eyes after using the personalized eye mask, as shown by the VISIA facial scanner ( Figure 5 I, J). By customizing personalized formulas and using DLP 3D printing technology to quickly prepare skin care products, it is possible to precisely treat individual skin problems, ensuring the accuracy and effectiveness of the treatment. In addition, this preparation method can quickly and conveniently provide users with skin care products that are completely tailored to their unique needs and preferences, which is beneficial for improving skin health and overall satisfaction. These results demonstrate that personalized skin care can be successfully achieved using new bio-inks and DLP 3D printers, thereby promoting the rapid development of the cosmetics industry.

[0087] Example 7 In vivo anti-aging effect of collagen eye mask

[0088] 1. Experimental process:

[0089] After a 7-day adaptation period, 24 male Kunming mice were subjected to short back hair shaving using an electric shaver, and then a depilatory cream was applied to remove the remaining hair, exposing a 4×4 cm area of ​​skin. The mice were randomly divided into 4 groups: control group (normal skin, no treatment), model group (aging skin, no treatment), CMA / Zn 2+ Group (aging skin, treated with CMA / Zn 2+ Eye mask treatment) and CMA / Zn 2+ / AHP-8 group (aging skin, treated with CMA / Zn 2+ / AHP-8 eye mask treatment). D-galactose solution (500 mg / kg / d) was subcutaneously injected for 28 consecutive days to induce skin aging in mice, and the control group was injected with 100 μL of 0.9wt% saline every day. 2+ Group and CMA / Zn 2+ The mice in the AHP-8 group were given the corresponding eye mask treatment every day. The body weight of the mice was recorded weekly to monitor their health status. The mice had free access to food and water throughout the experiment.

[0090] After 28 days of the experiment, the dermal density, transepidermal water loss (TEWL) and stratum corneum water content were measured using a composite probe to evaluate the anti-aging effect of the eye mask. The mice were killed and skin samples were taken from the treated areas. Half of the samples were fixed in 4% paraformaldehyde tissue fixative, paraffin sectioned, and stained with H&E and Masson's trichrome, followed by observation and photography under an optical microscope. The remaining half of the skin tissue homogenate was used to determine the relative content of hydroxyproline (HYP) in the skin using the chloramine-T colorimetric method, and the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were detected using a kit.

[0091] 2. Experimental results

[0092] To evaluate the efficacy of the printed eye mask in skin rejuvenation, we established an aging mouse skin model by subcutaneously injecting D-galactose ( Figure 6 A). D-galactose can induce oxidative stress and impair antioxidant defense mechanisms, and thus is used to simulate the natural aging process. 2+ / AHP-8) were applied to the back skin of mice every day, and CMA / Zn 2+ The hydrogel served as a treatment control group. Skin examination and histological analysis were performed 28 days after treatment. Mice in all groups showed consistent weight gain throughout the study, indicating their overall health ( Figure 6 B).

[0093] Combo probe was used for non-invasive measurement to assess skin aging. After 28 days, the skin was compared with the control group ( Figure 6 Compared with the model group, mice in the model group showed significant wrinkles ( Figure 6 Cb), proving that the induction of skin aging was successful. High-frequency ultrasound images showed that the dermal echo in the model group was weakened ( Figure 6 Db), indicating that aging leads to a decrease in collagen content and poor skin health. Quantitative analysis showed that the dermal density of the model group (34.30±2.11) was lower than that of the control group (61.96±4.39) ( Figure 6 E), which is consistent with the ultrasound image results. In contrast, the back skin of the treated mice was smoother and had fewer wrinkles, especially in the CMA / Zn 2+ / AHP-8 group( Figure 6 Cd). In addition, CMA / Zn 2+ and CMA / Zn 2+ Increased dermal echogenicity was observed in the AHP-8 group ( Figure 6 D), and the density of the latter ( Figure 6 Dd) is close to the control group ( Figure 6 Da). Quantitative analysis showed that the 2+ Compared with the model group, CMA / Zn 2+ The dermal density score was the highest in the AHP-8 group ( Figure 6 E), indicating that AHP-8 can effectively promote dermal regeneration.

[0094] The results of transepidermal water loss (TEWL) measurements showed that the TEWL in the model group increased, indicating that the skin barrier function was damaged after aging ( Figure 6 F) CMA / Zn 2+ Group and CMA / Zn 2+The TEWL value of the / AHP-8 group was significantly lower than that of the model group, and there was no significant difference between the latter and the control group, indicating that the printed eye mask has the effect of preventing transepidermal water loss. In addition, the skin water content measurement results showed that the CMA / Zn 2+ The water content of the / AHP-8 group (121.21±8.65) was significantly higher than that of the model group (90.80±5.06) and the CMA / Zn 2+ Group (104.10±4.12) Figure 6 G), indicating CMA / Zn 2+ / AHP-8 eye mask has better moisturizing effect. These results show that the new CMA / Zn 2+ The bioink was able to successfully deliver anti-wrinkle peptides, demonstrating the effectiveness of personalized eye masks in anti-aging.

[0095] To further evaluate the effect of the anti-aging eye mask, we performed histological analysis using H&E and Masson's trichrome staining ( Figure 7 A). The staining results showed that the skin structure of the control mice was intact, the dermis was thicker, and it was closely connected to the intact epidermis, which was normal and healthy skin. The dermis tissue structure of the model group was disordered and broken, which may be related to aging. In addition, the collagen fiber breakage observed in the aging skin model may be the cause of skin wrinkles.

[0096] CMA / Zn 2+ Aged skin treated with CMA / Zn / AHP-8 eye mask showed stronger anti-aging effects, characterized by a denser skin structure and more orderly collagen fiber arrangement, similar to normal skin. 2+ It also showed an effect of improving healing, but the effect was not as significant as that observed with the eye mask containing anti-wrinkle peptides. Quantitative analysis further found that ( Figure 7 B), CMA / Zn 2+ The collagen deposition in the / AHP-8 group (65.07%) was significantly higher than that in the model group (38.91%) and the CMA / Zn 2+ The results showed that the eye mask has a significant ability to promote collagen regeneration in the 20% to 30% group (51.64%), indicating that the eye mask has a significant ability to promote collagen regeneration. Importantly, this value is not significantly different from normal skin (67.67%), showing the effectiveness of the eye mask in resisting skin aging. These findings are consistent with previous studies that AHP-8 has good anti-aging effects.

[0097] In order to explore the CMA / Zn 2+ / AHP-8 eye mask's anti-aging mechanism, we detected the levels of hydroxyproline (HYP), superoxide dismutase (SOD), and malondialdehyde (MDA) in the skin. HYP is a unique amino acid in collagen. HYP can be used as an anti-aging indicator to quantitatively analyze the content of collagen in the skin. The content of HYP in each group ( Figure 7 C) corresponds to the results of collagen deposition and dermal density, further emphasizing the superior ability of the printed eye mask in promoting collagen synthesis. SOD is an antioxidant enzyme that catalyzes the decomposition of superoxide anions into hydrogen peroxide and oxygen, and MDA is the final product of lipid peroxidation. 2+ In the / AHP-8 group, SOD levels were significantly increased ( Figure 7 D), the expression of MDA was significantly decreased ( Figure 7 E), indicating that the eye mask has excellent antioxidant capacity. 2+ / AHP-8 eye mask may fight skin aging by reducing oxidative stress and promoting collagen regeneration.

[0098] In summary, the present invention provides a Zn 2+ DLP 3D printing bio-ink coordinated with collagen, the bio-ink comprising the following components: 0.1-1% m / v methacrylated collagen solution, 0.1-0.5% m / v photoinitiator, 0-0.5% m / v UV absorber and 0.01-0.1% m / v Zn 2+ Solution. The bio-ink uses a method combining metal ion coordination and chemical cross-linking to prepare a hydrogel with high mechanical properties, strong anti-degradation ability and good biocompatibility, and the preparation conditions are simple and mild, and the complete triple helix structure and biological activity of collagen are retained to the greatest extent; the bio-ink of the present invention uses DLP 3D printing technology to customize skin care products, overcoming the defect that popular skin care products cannot meet individual needs; compared with ordinary skin care products using base cloth as a carrier, collagen is used as a hydrogel carrier to extend the application time and has a more lasting moisturizing and anti-aging ability; at the same time, after adding the active ingredient acetyl hexapeptide-8, dynamic lines, static lines and fine lines can be improved, and the synthesis of collagen, elastin and hyaluronic acid can be promoted, which has the effect of tightening the skin.

[0099] The above description is only the details of individual exemplary implementation cases of the present invention. For those skilled in the art, the present invention may have various modifications and changes according to specific preparation conditions during actual application, and is not intended to limit the present invention. Anything within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Zn 2+ DLP 3D printing bio-ink with collagen, characterized in that: The bio-ink comprises the following components: 0.1-1% m / v methacrylated collagen solution, 0.1-0.5% m / v photoinitiator, 0.05-0.5% m / v UV absorber and 0.01-0.1% m / v Zn 2+ Solution.

2. The bio-ink according to claim 1, characterized in that: The bio-ink comprises the following components: 0.5-1% m / v methacrylated collagen solution, 0.25-0.5% m / v photoinitiator, 0.1-0.5% m / v UV absorber and 0.01% m / v Zn 2+ Solution.

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

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

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

6. The bio-ink according to claim 1, characterized in that: The ultraviolet absorber includes one or more of UA, lemon yellow and food coloring brilliant blue.

7. The method for preparing the biological ink according to any one of claims 1 to 6, characterized in that: The steps include: (1) dissolving freeze-dried collagen sponge in acetic acid solution to prepare collagen solution, and slowly stirring to dissolve; (2) adjusting the pH of the collagen solution obtained in step (1) to 7-9, adding methacrylic anhydride dropwise, and maintaining the pH of the solution at 7-8; reacting for 24 hours under ice bath conditions, dialyzing, and freeze-drying to obtain methacrylated collagen; (3) Prepare 0.1-1% m / v methacrylated collagen solution with 0.5M acetic acid solution, add 6M sodium hydroxide solution to adjust the pH of the solution to neutral, add 0.1-0.5% m / v photoinitiator, 0-0.5% m / v UV absorber and 0.01-0.1% m / v Zn 2+ The solution was mixed evenly and centrifuged to remove bubbles to obtain CMA / Zn 2+ Bio-ink.

8. The preparation method according to claim 7, characterized in that: The pH of the collagen solution in steps (2) and (3) is adjusted using sodium hydroxide solution.

9. A skin repair material, characterized in that: The skin repair material is obtained by adding active ingredients to the biological ink described in any one of claims 1 to 6 and printing it through DLP 3D printing.

10. Use of the bio-ink as described in any one of claims 1 to 6 in the preparation of skin repair materials, bone-cartilage repair materials, and tissue engineering.

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