Collagen DLP 3D printing bio-ink for personalized skin care as well as preparation method and application of collagen DLP 3D printing bio-ink

Abstract

The invention belongs to the technical field of biology, and relates to collagen DLP 3D printing bio-ink for personalized skin care, a preparation method and application, and the collagen DLP 3D printing 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 0.00001%-0.001% m / v of 1, 4-butanediol glycidyl ether. The bio-ink disclosed by the invention can realize high-precision DLP 3D printing, and collagen gel obtained by printing has enhanced mechanical properties and compression resistance; meanwhile, by adding the active ingredients, the skin texture can be enhanced, fine wrinkles are reduced, the thickness of epidermis and dermis is increased, and skin repair is promoted.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a collagen DLP 3D printing bio-ink for personalized skin care, a preparation method and an application thereof. Technical Background

[0002] As an innovative manufacturing technology, 3D bioprinting can accurately position and assemble biomaterials to manufacture biomedical products with complex shapes through computer-aided design (CAD) and numerical control systems. This technology has been widely used in the fields of regenerative medicine and tissue engineering due to its fast, accurate and highly personalized characteristics. However, traditional inkjet and extrusion 3D bioprinting technologies face some challenges in practical applications: inkjet printing is prone to nozzle clogging due to the use of high-viscosity materials, while extrusion printing is limited by its long production time and low printing accuracy.

[0003] In contrast, digital light processing (DLP) bioprinting technology has shown significant potential in disease modeling and regenerative medicine due to its rapid and high-precision characteristics. Nevertheless, the core of DLP printing technology - the preparation of bio-ink - still faces challenges. The ideal bio-ink should have fast curing, easy control, good biocompatibility, mechanical stability, biodegradability and biomimetic properties. Although gelatin is widely used as a common bio-ink component, its insufficient biological activity limits its prospects in clinical applications.

[0004] As the largest organ in the human body, the skin plays an important role in defending against external aggressions. Its health is affected by many factors, such as ultraviolet radiation, environmental toxins, and mechanical damage. Faced with the existing anti-aging products on the market, the cosmetics industry is still seeking more effective and personalized solutions. For example, leading companies such as Neutrogena, AmorePacific, and Procter & Gamble (P&G) have adopted 3D printing technology to customize products according to consumers' specific facial features and skin care needs.

[0005] As a key component of the skin, collagen is often added to facial masks and other skin care products due to its role in promoting cell proliferation, differentiation and migration. However, the collagen-based bio-ink currently on the market has deficiencies in mechanical properties, which limits its application in personalized DLP 3D printing technology and makes it difficult to meet the demand for personalized skin care solutions. Summary of the invention

[0006] In view of the above problems, the object of the present invention is to provide a collagen DLP 3D printing bio-ink for personalized skin care, wherein the bio-ink comprises the following components: 0.1% to 1% m / v methacrylated collagen solution, 0.1% to 0.5% m / v photoinitiator, 0.05% to 0.5% m / v UV absorber and 0.00001%-0.001% m / v 1,4-butanediol glycidyl ether.

[0007] Preferably, the bio-ink comprises the following components: 1% m / v methacrylated collagen solution, 0.1% to 0.5% m / v photoinitiator, 0.05% to 0.5% m / v UV absorber and 0.00001% to 0.001% m / v 1,4-butanediol glycidyl ether.

[0008] Preferably, the collagen is yak collagen.

[0009] Preferably, the yak collagen is type I yak collagen, and / or type II yak collagen, and / or type II yak 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 one or more of UA, lemon yellow, and food coloring brilliant blue.

[0013] Preferably, the ultraviolet absorber is UA.

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

[0015] (1) dissolving freeze-dried collagen in acetic acid, stirring until completely dissolved, adding methacrylic anhydride dropwise to the collagen solution, maintaining the pH of the solution at a weak alkaline state during the reaction, reacting at a low temperature, dialyzing, and freeze-drying to obtain methacrylated collagen;

[0016] (2) adding the methacrylated collagen obtained in step (1) to an acetic acid solution, stirring until completely dissolved, and adjusting the pH to neutral to obtain a methacrylated collagen solution;

[0017] (3) Taking the methacrylated collagen solution obtained in step (2) at a concentration of 0.1% to 1% m / v, adding 0.1% to 0.5% m / v photoinitiator, 0.05% to 0.5% m / v UV absorber and 0.00001% to 0.001% m / v 1,4-butanediol glycidyl ether, vortexing to mix evenly, and obtaining a bio-ink.

[0018] Preferably, sodium hydroxide solution is used to adjust the pH.

[0019] 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 and printing it through DLP 3D.

[0020] The fourth object of the present invention is to provide the use of the biological ink in the preparation of skin repair materials.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention provides a collagen DLP 3D printing bio-ink for personalized skin care, wherein the bio-ink comprises the following components: 0.1% to 1% m / v methacrylated collagen solution, 0.1% to 0.5% m / v photoinitiator, 0.05% to 0.5% m / v UV absorber and 0.00001%-0.001% m / v 1,4-butanediol glycidyl ether.

[0023] (2) The bio-ink of the present invention can realize high-precision DLP 3D printing; and the bio-ink of the present invention adds the photoinitiator and 1,4-butanediol glycidyl ether at the same time, so that the printed collagen gel has more stable mechanical properties and thermal stability; while the existing bio-ink is immersed in chemical cross-linking after printing, and the chemical cross-linking agent is located on the surface of the material, resulting in the disadvantages of complete external cross-linking, incomplete internal cross-linking, and unstable material;

[0024] (3) The bio-ink of the present invention has simple and mild preparation conditions, retains the complete triple helix structure of collagen to the greatest extent, is conducive to cell survival and adhesion, and successfully realizes bio-printing with loaded cells;

[0025] (4) Collagen has skin care effects such as moisturizing, locking in moisture, and anti-wrinkle, and has good biocompatibility and is gentle and non-irritating to the skin. At the same time, adding the active ingredient retinol can enhance skin texture, reduce fine lines, increase the thickness of the epidermis and dermis, and promote skin repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Effect of BDDE concentration in the CMA / BDDE bio-ink described in the present invention.

[0027] Note: (A) Strain sweep test graph of CMA and CMA / BDDE gel and (B) compression curve graph. (C) Cell viability graph of cell-laden gel on day 1 and day 7 of culture and (D) Live / dead cell staining graph on day 1, day 4, and day 7.

[0028] Figure 2 Printability of the CMA / BDDE bio-ink described in the present invention.

[0029] Note: (A) (a) cat's paw, (b) hollow hexagonal prism, (c) 3D model of hollow complex structure and (B) actual printed images at different angles.

[0030] Figure 3 The cell-carrying printing capability of the CMA / BDDE bio-ink described in the present invention.

[0031] Note: (A) Actual photos of cell-loaded CMA / BDDE bio-ink prints in different shapes such as five-pointed stars, leaves, spades, diamonds, and plum blossoms. (B) Live / dead cell staining, (C) bright field photography, and (D) quantitative analysis of cell-loaded CMA / BDDE printed grids. (E) Cell adhesion skeleton staining of CMA and CMA / BDDE cell-loaded printed grids.

[0032] Figure 4 The personalized skin care strategy represented by the eye mask described in the present invention.

[0033] Note: (A) Schematic diagram of the personalized skin care process. (B) 3D eye mask model designed based on human face. (C) Printing ink with active ingredient retinol. (D) DLP 3D printed eye mask and properties (a) DLP 3D printed (b) hydrogel eye mask and (c) adhesion test on fingers and gloves. (E) Eye skin status on day 0, day 7, and day 14.

[0034] Figure 5 Evaluation of the efficacy of the personalized eye mask of the present invention on an animal model.

[0035] Note: (A) Schematic diagram of the experimental process. (B) Dermoscopy of the control group, model group, CMA / BDDE group, and CMA / BDDE / VA group, (C) ultrasound skin detection image, (D) skin density, (E) transepidermal water loss, and (F) hydration.

[0036] Figure 6 Histological analysis of the mouse anti-aging model described in the present invention.

[0037] Note: (A) H&E staining of the control group, model group, CMA / BDDE group and CMA / BDDE / VA group, (B) Masson staining, (C) collagen deposition analysis, (D) HYP, (E) SOD and (F) MDA content detection. Specific implementation methods

[0039] 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.

[0040] It should be noted that, in the following examples, unless otherwise specified, the methods used are all conventional methods; the reagents used are all conventional reagents and can be purchased from the market.

[0041] In the embodiment of the present invention, yak type I collagen (for the extraction method, see invention patent CN113520900B) is used as a raw material 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.

[0042] In the following embodiments, the collagen DLP 3D printing bio-ink is called bio-ink before printing and is called hydrogel after printing.

[0043] Example 1 Preparation of collagen DLP 3D printing bio-ink

[0044] 300 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. 1 mL of methacrylic anhydride (MA) was added dropwise to the dissolved collagen solution, and NaOH was added during the reaction to keep the pH of the solution weakly alkaline. After 24 hours of low-temperature reaction, ultrapure water was used for dialysis to remove residual MA. The resulting methacrylated collagen (CMA) was freeze-dried and stored at -20 °C for further use.

[0045] 500 mg of CMA solid was added to 0.5M 50 mL acetic acid solution and stirred at 4°C until completely dissolved, and then NaOH solution was added to adjust the pH to neutral. In the completely dissolved CMA solution, photoinitiator (LAP) solution, ultraviolet light absorber (UA) solution and 1,4-butanediol glycidyl ether (BDDE) solution were added, and vortexed to mix evenly to obtain CMA / BDDE bio-ink.

[0046] In order to determine the effects of CMA solution concentration, photoinitiator LAP concentration, UV absorber UA concentration and crosslinker BDDE on 3D printing bio-ink, we prepared CMA / BDDE bio-ink according to Table 1. All the bio-inks prepared were uniform in texture and no flocculent precipitation was produced.

[0047] Among them, bio-inks 1, 2, 3, and 4 have low collagen concentrations and poor mechanical properties, and cannot form gels with specific shapes; bio-inks 5, 6, 7, 8, 9, and 10 have moderate fluidity and high printing precision, but bio-inks 5 and 6 require a long printing time, bio-ink 10 has low printing precision, and high concentrations of LAP have certain cytotoxicity. Therefore, we selected bio-inks 7, 8, and 9 as printing inks for subsequent experiments.

[0048] Table 1 Content of each component in different bio-inks

[0049]

[0050]

[0051] Example 2 Effect of BDDE concentration in collagen DLP 3D printing bio-ink

[0052] 1. Experimental Procedure

[0053] Amplitude sweep tests were used to detect the storage modulus (G') and loss modulus (G") of the gel under different strain conditions. The ink to be tested was added to a cylindrical mold and cured into a gel under UV light. The cured gel was taken out of the mold and placed under the probe of the rheometer. The angular frequency was set to a constant 1 rad / s, and then the G' and G" of the gel were measured in the strain range of 0.1%-100%.

[0054] The stress-strain curves of CMA and CMA / BDDE gels were measured on a universal testing machine. 2.5 mm × 13 mm cylindrical gels were prepared as above, and then the samples were subjected to compression tests at a constant speed of 15 mm / min.

[0055] CCK-8 assay was used to detect cytotoxicity. 100 μL of cell-loaded bio-ink was pipetted into a 96-well plate and cured under a 405 nm UV lamp for 30 s. Subsequently, 200 μL of complete culture medium was added to the 96-well plate and incubated in a 5% CO2, 37°C incubator. On the 1st, 4th, and 7th day of incubation, CCK-8 solution was added and the OD value at 450 nm was measured using a microplate reader.

[0056] Live / dead cell staining experiments were used to determine the compatibility of bio-inks. 100 μL of cell suspension was added to bio-inks 7, 8, and 9, and gently shaken to further mix. 250 μL of the above bio-inks were pipetted into a confocal dish (15 mm in diameter) and cured for 30 seconds under 405 nm UV light. The gel was then washed three times with complete culture medium to remove uncured bio-ink on the surface. Finally, 2 mL of complete culture medium was added to the confocal dish and cultured in a 5% CO2, 37°C incubator. Calcein-AM and propidium iodide (PI) were used to stain the gel on the first and seventh days of culture, and images of the gel interior were collected by laser confocal microscopy, in which live cells showed yellow-green fluorescence and dead cells showed red fluorescence.

[0057] 2. Experimental results

[0058] In order to evaluate the effect of BDDE concentration on the rheological properties of collagen gels, strain sweep tests were performed on the bio-ink CMA and the cured gels of bio-inks 7, 8, and 9 (CMA, CMA / BDDE-1, CMA / BDDE-2, and CMA / BDDE-3). Figure 1 A). The experimental results show that the gel exhibits excellent elastic-gel properties in the low strain region with G'>G", until the shear strain increases to 10%. It is worth noting that the storage modulus of CMA / BDDE gel is greater than that of CMA gel and increases with the increase of BDDE concentration.

[0059] In addition, compression experiments were performed to further evaluate the response of BDDE concentration to mechanical stress in collagen gels ( Figure 1 B). As the strain increases, the compressive stress of the gel increases gradually, and the compressive stress of the CMA / BDDE-3 group is the largest, reaching 30.90 kPa. These results show that as the concentration of BDDE increases, the compressive strength of the gel also increases.

[0060] High biocompatibility is a prerequisite for cell-laden bioprinting. Therefore, we studied the effect of gel on the viability and distribution of encapsulated cells by CCK-8 assay and live / dead cell staining. First, L929 cells were mixed with CMA / BDDE to prepare bio-ink, and then the gel was prepared by UV curing under sterile conditions. Another equal amount of L929 cells was mixed with CMA as a control group. After 7 days of culture, there was no significant change in cell viability, and the cell viability in CMA / BDDE-3 and CMA gels was 95.68±0.71% and 98.25±0.43%, respectively ( Figure 1 C). In live / dead cell staining, gels containing 3 different concentrations of BDDE showed no significant cytotoxicity to encapsulated cells within 7 days ( Figure 1D). These results highlight the excellent biocompatibility of CMA / BDDE gel and indicate that this bio-ink is suitable for 3D printing with cells. Therefore, we selected bio-ink 9 as the printing ink for subsequent experiments.

[0061] Example 3 Printability of collagen DLP 3D printing bio-ink

[0062] 1. Experimental Procedure

[0063] Add bubble-free CMA / BDDE bio-ink to the DLP 3D printer’s cartridge. Set basic printing parameters as follows: layer height 100 μm, light intensity 15 mW / cm 2 , 6 base layers, 24s exposure time for base layer, 19s exposure time for sheet layer. After printing, the printed gel model was gently scraped off the deposition platform, and the surface of the gel was gently rinsed with ultrapure water to remove the uncured ink adhering to the surface. Then, secondary curing was performed under ultraviolet light to obtain the CMA / BDDE hydrogel of the preset shape.

[0064] 2. Experimental results

[0065] CMA / BDDE bio-ink has UV curing properties and can be precisely 3D printed through digital light processing (DLP), thereby producing gel structures with high resolution and complex geometries. Figure 2 As shown in the figure, we successfully prepared a variety of objects such as cat's paws, hollow hexagonal prisms, and hollowed-out complex structures. It is worth noting that the printed cat's paw structure has a flat surface and the hollow hexagonal prism has a clear edge. In addition, the hollowed-out complex structure also shows intricate details and excellent support. These results show that CMA / BDDE bioink can achieve ultra-high resolution, good printing accuracy, and excellent fidelity in building complex structures that have rarely been achieved before.

[0066] Example 4: Cell-loaded printing of collagen DLP 3D printed bio-ink

[0067] 1. Experimental Procedure

[0068] The steps for printing cell-loaded CMA and CMA / BDDE and live / dead cell staining are as follows: 3 mL of bio-ink was added to the sterilized DLP 3D printer tank and printed according to the printing parameters described above. The printed gel was carefully washed three times in complete culture medium and transferred to a confocal culture dish containing 2 mL of complete culture medium for incubation. It was then stained as described above and imaged using a laser confocal microscope.

[0069] The steps of cell adhesion and skeleton staining of printed CMA and CMA / BDDE hydrogels are as follows: digested L929 cells are used to prepare a high-concentration cell suspension using complete culture medium, and then added to the confocal dish containing 3D-printed CMA / BDDE hydrogels. After incubation in a 5% CO2, 37°C incubator for 24 hours, the upper culture medium is aspirated. The cytoplasm is stained with phalloidin, and the nucleus is stained with Hoechst 33342. The stained gel is then placed under a laser confocal microscope for observation.

[0070] 2. Experimental results

[0071] To illustrate the cell-carrying printing capability of CMA / BDDE bio-ink, we successfully printed various complex models. Stars, leaves, spades, diamonds, and plum blossoms loaded with L929 cells were successfully printed ( Figure 3 A). Among them, the grid model was specially selected for staining observation to confirm the distribution of cells in the CMA / BDDE hydrogel during printing and culture. It is worth noting that the cells in the CMA / BDDE hydrogel are evenly dispersed on the scaffold ( Figure 3 B), and the connection of two vertical filaments in the 3D mesh shows good interconnectivity ( Figure 3 C), there is no significant difference between the pore sizes ( Figure 3 D). HFF-1 cells were cultured on CMA and CMA / BDDE hydrogels, and the cytoskeleton was observed by staining actin and nuclei. After 24 h of culture, the cells adhered to the scaffold surface and spread well, showing a characteristic spindle shape ( Figure 3 E). These results suggest that CMA / BDDE bioink has the potential to be applied in personalized drug delivery therapy.

[0072] Example 5 Personalized skin care strategy represented by eye mask

[0073] 1. Experimental Procedure

[0074] A digital camera was used to take photos of the face information, and the photographed face was 3D modeled using Blender 3.5 software, and an eye mask with a specific size and shape was designed based on the modeled face. In order to further enhance the anti-aging effect of CMA / BDDE bio-ink, 100μL of 0.5% retinol (VA) solution was added to the above CMA / BDDE bio-ink. Subsequently, DLP 3D printing technology was used to print personalized eye masks containing the active ingredient VA, and the eye masks were applied to the eyes of volunteers every day. On the 1st, 7th and 14th days, the skin condition was monitored using the VISIA skin detector.

[0075] 2. Experimental results

[0076] To verify the potential of CMA / BDDE bio-ink in personalized skin care, we used DLP 3D printing technology to prepare a collagen eye mask that meets the anti-wrinkle needs of volunteers ( Figure 4 A). First, by scanning the facial contour and facial physiological features, an eye mask that fits the individual's facial size is designed ( Figure 4 B). Topical retinol (VA) has been shown to enhance skin texture, reduce fine lines, and increase the thickness of the epidermis and dermis. Therefore, VA was introduced into CMA / BDDE bio-ink as a personalized pharmaceutical ingredient for anti-wrinkle ( Figure 4 C). Subsequently, the CMA / BDDE / VA hydrogel was successfully printed, showing good adhesion to fingers and gloves and being able to adapt to various hand movements ( Figure 4 D). After applying the hydrogel, the skin condition was tested using the VISIA skin detector on days 0, 7, and 14 ( Figure 4 E). The data results showed that the number and area of ​​eye wrinkles of volunteers gradually decreased, with wrinkle scores of 18.481, 14.612, and 10.314, respectively. In addition to retinol used in this study, other functional ingredients such as niacinamide and salicylic acid can also be added to meet the needs of users for whitening, exfoliation, and other issues. These adhesion properties and wrinkle reduction effects show the potential of the gel as a customizable dressing for personalized medicine.

[0077] Example 6 In vivo anti-aging effect of personalized collagen eye mask

[0078] 1. Experimental process:

[0079] After 24 Kunming mice (male) were adapted to feeding for 1 week, a 4×4 cm area was taken from the back of their necks, and the hair on the back was removed by electric shovel, and then the remaining hair was removed by depilatory cream. Subsequently, the depilatory mice were randomly divided into 4 groups, with 6 mice in each group: control group, model group, CMA / BDDE group and CMA / BDDE / VA group. The control group mice were injected with 200 μL of 0.9wt% saline on the back every day, and the other groups of mice were injected with 200 μL of D-galactose solution on the back every day, with a dose of 500 mg / kg / d. At the same time as modeling, the treatment group was given corresponding treatment, and the CMA / BDDE group and CMA / BDDE / VA group were treated with their own gels every day. During the administration period, the physical signs of mice in each group were continuously observed and recorded, such as: changes in mouse weight, drinking water and diet, skin color, etc. In addition, the skin of the depilatory area of ​​the mice was tested by dermoscopy, high-frequency ultrasound, transepidermal water loss, stratum corneum water content, dermal density, etc. using a Combo instrument. The experiment lasted for four weeks, and the mice were euthanized after the last administration. During the feeding period, all mice were free to take feed and drink water. The specific route and method of administration are shown in the table below.

[0080] Table 2 Specific routes and methods of administration

[0081]

[0082] Subsequently, part of the mouse back skin was subjected to H&E staining and Masson staining, and part of the skin was quantified by the chloramine T method to quantify the content of hydroxyproline in the skin, and the content of superoxide dismutase (SOD) and malondialdehyde (MDA) in the skin was quantified by a kit.

[0083] 2. Experimental results

[0084] To further study the effectiveness of CMA / BDDE / VA in vivo, we established a D-galactose-induced aging mouse model for anti-aging treatment. The mice were randomly divided into 4 groups: control group, model group, CMA / BDDE group, and CMA / BDDE / VA group. Among them, the mice in the model group, CMA / BDDE group, and CMA / BDDE / VA group were subcutaneously injected with D-galactose solution on the back every day, while the mice in the control group were injected with an equal amount of normal saline every day. Subsequently, the mice in the treatment group were treated with gel patches of CMA / BDDE and CMA / BDDE / VA, respectively, and the mouse skin was instrumentally detected and histologically analyzed after 28 days. ( Figure 5 A). Four weeks after modeling, obvious wrinkles appeared on the back skin of mice in the model group, indicating that the modeling was successful ( Figure 5 B). Compared with the model group, the echogenicity of the CMA / BDDE / VA group was significantly enhanced and the dermal density was significantly improved ( Figure 5 C). Dermal strength is related to the content of structural proteins such as collagen and elastin in the skin. The CMA / BDDE / VA group showed increased dermal strength, increased structural protein content, and better anti-aging ability ( Figure 5 D). Changes in transepidermal water loss (TEWL) and hydration were measured to assess disruption of the skin's water barrier function. TEWL decreased in all treatment groups ( Figure 5 E), hydration increased ( Figure 5 F), indicating that the dressing has the functions of replenishing water, moisturizing and repairing the skin barrier.

[0085] Mouse skin histological examination further verified the anti-aging mechanism of CMA / BDDE and CMA / BDDE / VA gels ( Figure 6A). Hematoxylin-eosin (H&E) staining results showed that the control group mice had healthy skin characteristics, intact epidermal tissue, thick dermis, and clear epidermal-dermal connection. In the model group, the epidermis was unevenly thick and the dermis structure was destroyed. After treatment with CMA / BDDE / VA, the aging state was alleviated, and the treatment effect was better than that of the CMA / BDDE group. Aniline blue dye can effectively dye collagen fibers blue. Masson staining results showed that the collagen fibers in the skin of control group mice were wavy and densely arranged, while the collagen fibers in the aging model group were disordered, broken, and loose. At the same time, the repair effect of the CMA / BDDE / VA group was better than that of the CMA / BDDE group, and the collagen fibers were denser and richer. Reduced collagen levels in the skin can lead to wrinkles and other aging characteristics. Determination of skin collagen deposition and hydroxyproline (HYP) concentration can help quantitatively analyze the collagen content in the skin. The results show that CMA / BDDE / VA can promote collagen deposition ( Figure 6 B), increasing the content of hydroxyproline, showing enhanced anti-aging effects ( Figure 6 C). Malondialdehyde (MDA) is the end product of lipid peroxidation, while superoxide dismutase (SOD) is an essential antioxidant enzyme for the body. The content of MDA and SOD indirectly reflects the degree of lipid peroxidation. The experimental results show that CMA / BDDE / VA has a significant antioxidant effect, which is manifested by promoting the secretion of SOD ( Figure 6 D) and inhibiting MDA secretion ( Figure 6 E). CMA / BDDE / VA has moisturizing, antioxidant and anti-aging effects in mice.

[0086] In summary, the present invention provides a collagen DLP 3D printing bio-ink for personalized skin care, wherein the bio-ink comprises the following components: 0.1% to 1% m / v methacrylated collagen solution, 0.1% to 0.5% m / v photoinitiator, 0.05% to 0.5% m / v UV absorber and 0.00001% to 0.001% m / v 1,4-butanediol glycidyl ether. The bio-ink described in the present invention can realize high-precision DLP 3D printing; and the bio-ink described in the present invention, by adding a photoinitiator and BDDE at the same time, the prepared collagen gel has enhanced mechanical properties and compression resistance; while the existing bio-ink is immersed in chemical cross-linking only after printing, and the chemical cross-linking agent is located on the surface of the material, resulting in the disadvantages of complete external cross-linking, incomplete internal cross-linking, and unstable material; the bio-ink described in the present invention has simple and mild preparation conditions, retains the complete triple helix structure of collagen to the greatest extent, is conducive to cell survival and adhesion, and can realize 3D printing of loaded cells; collagen has skin care effects such as moisturizing, water locking, and anti-wrinkle, and has good biocompatibility and is mild and non-irritating to the skin; at the same time, adding the active ingredient retinol can enhance skin texture, reduce fine lines, increase the thickness of the epidermis and dermis, and promote skin repair.

[0087] 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 collagen DLP 3D printing bio-ink for personalized skin care, characterized in that: The biological ink comprises the following components: 0.1% to 1% m / v methacrylated collagen solution, 0.1% to 0.5% m / v photoinitiator, 0.05% to 0.5% m / v ultraviolet absorber and 0.00001% to 0.001% m / v 1,4-butanediol glycidyl ether.

2. The bio-ink according to claim 1, characterized in that: The biological ink comprises the following components: 1% m / v methacrylated collagen solution, 0.1%-0.5% m / v photoinitiator, 0.05%-0.5% m / v ultraviolet absorber and 0.0001%-0.001% m / v 1,4-butanediol glycidyl ether.

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

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

5. The bio-ink according to claim 2, characterized in that: The photoinitiator includes one or more of LAP, TPO and I2959.

6. The bio-ink according to claim 2, characterized in that: The ultraviolet absorber includes one or more of UA, lemon yellow and food coloring brilliant blue.

7. The method for preparing a biological ink according to any one of claims 1 to 6, characterized in that: The steps include: (1) dissolving freeze-dried collagen in acetic acid, stirring until completely dissolved, adding methacrylic anhydride dropwise to the collagen solution, maintaining the pH of the solution at a weak alkaline state during the reaction, reacting at a low temperature, dialyzing, and freeze-drying to obtain methacrylated collagen; (2) adding the methacrylated collagen obtained in step (1) to an acetic acid solution, stirring until completely dissolved, and adjusting the pH to neutral to obtain a methacrylated collagen solution; (3) Taking the methacrylated collagen solution obtained in step (2) at a ratio of 0.1% to 1% m / v, adding 0.1% to 0.5% m / v photoinitiator, 0.05% to 0.5% m / v UV absorber and 0.00001% to 0.001% m / v 1,4-butanediol glycidyl ether, vortexing to mix evenly, to obtain bio-ink.

8. The preparation method according to claim 7, characterized in that: Sodium hydroxide solution was used to adjust pH.

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 biological ink according to any one of claims 1 to 6 in preparing skin repair materials.

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