An inorganic / organic composite micro-patterned multicellular scaffold and its preparation method and application

Abstract

The present invention relates to an inorganic / organic composite micro-patterned multicellular scaffold, its preparation method, and application. The inorganic / organic composite micro-patterned multicellular scaffold comprises a three-dimensional network-like scaffold framework formed by arranging inorganic / organic composite bio-ink loaded with cells A, and a scattered-point framework filled with hydrogel bio-ink loaded with cells B within the pore structure of the three-dimensional network-like scaffold framework.

Description

Technical Field

[0001] The present invention relates to an inorganic / organic composite micro-patterned multicellular scaffold and its preparation method and application, and specifically to a complex micro-patterned scaffold in which multiple cells are distributed in a network-like and point-like manner in three-dimensional space and its preparation method and application, belonging to the field of biotechnology. Background Art

[0002] Hair loss caused by hair follicle loss and degeneration seriously impacts a person's physical and mental health. As a crucial appendage of skin tissue, hair follicles play a vital role in hair growth, thermoregulation, secretion, and excretion. Furthermore, hair follicle stem cells within hair follicles participate in wound contraction, angiogenesis, and skin re-epithelialization. However, adult hair follicles cannot regenerate after loss or necrosis. Therefore, hair follicle regeneration has become a key area of ​​research in skin tissue engineering. Studies have shown that the growth activity of hair follicles is closely linked to the surrounding dermal vascular network. Therefore, skin substitutes that effectively integrate the complex structure and biological functions of hair follicles and blood vessels are of great significance for hair regeneration and functional skin regeneration.

[0003] In recent years, cell patterning has emerged to promote the occurrence of cell behavior and cell communication necessary for tissue regeneration. In order to establish a bioactive microenvironment with spatial patterns of multiple cell types, three-dimensional (3D) bioprinting has shown great application potential in the preparation of biomimetic artificial skin substitutes. As an emerging additive manufacturing technology, bio-3D printing technology can precisely control the spatial distribution of biomaterials, living cells and growth factors. However, existing bio-3D printed skin tissue engineering scaffolds have difficulty integrating the biomimetic structure and biological function of hair follicles and vascular networks. Therefore, biomimetic hair follicle-vascular bio-3D printed skin substitutes that can be used to induce hair regeneration and hair follicle reconstruction have become an urgent need to achieve functional skin tissue regeneration. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides an inorganic / organic composite micro-patterned multicellular scaffold and its preparation method and application.

[0005] On the one hand, the present invention provides an inorganic / organic composite micropatterned multicellular scaffold, comprising: a three-dimensional network-like scaffold framework formed by the arrangement of inorganic / organic composite bio-ink loaded with cells A, and a scattered point-like framework formed by the arrangement of hydrogel bio-ink loaded with cells B filled in the pore structure of the three-dimensional network-like scaffold framework.

[0006] In the present invention, the inorganic / organic composite micropatterned multicellular scaffold simulates the dense vascular network and hair follicle structure distributed in human skin tissue, can induce hair regeneration and vascular reconstruction, and is of great significance for skin tissue repair, skin appendage regeneration and skin physiological function recovery.

[0007] Preferably, the three-dimensional network-like scaffold framework formed by the inorganic / organic composite bio-ink loaded with cells A has a porosity of 30-50%, and a diameter of the pore structure is 800 μm-2 mm.

[0008] Preferably, the three-dimensional network-shaped stent framework and the scattered point-shaped framework form a bionic pattern.

[0009] Preferably, the inorganic / organic composite bio-ink loaded with cells A comprises cells A, a bioactive inorganic material and a first hydrogel matrix;

[0010] Preferably, the cell A is a vascular endothelial cell, preferably one of a human umbilical vein endothelial cell and a human dermal microvascular endothelial cell;

[0011] Preferably, the bioactive inorganic material is magnesium silicate hollow nanospheres, and more preferably, the particle size of the magnesium silicate hollow nanospheres is 400 to 600 nm.

[0012] Preferably, the cell B-loaded hydrogel bio-ink comprises cells B and a second hydrogel matrix;

[0013] Preferably, the cell B is a dermal papilla cell, preferably one of a human dermal hair papilla cell and a human hair follicle stem cell; the loading amount of the cell B in the hydrogel bio-ink loaded with the cell B is 3 to 5 million / mL.

[0014] Preferably, the first hydrogel matrix comprises at least one of methacrylated gelatin, methacrylated hyaluronic acid, collagen, and methylcellulose, preferably a uniform mixture of methacrylated gelatin and methylcellulose; more preferably, the mass ratio of methacrylated gelatin to methylcellulose in the uniform mixture of methacrylated gelatin and methylcellulose is (100-150):1.

[0015] Preferably, the second hydrogel matrix comprises at least one of methacrylated gelatin, methacrylated hyaluronic acid, collagen, and methylcellulose, preferably methacrylated gelatin.

[0016] Preferably, the mass of the bioactive inorganic material does not exceed 8 wt % of the mass of the first hydrogel matrix, preferably 0.1 to 6 wt %;

[0017] The loading amount of cells A in the inorganic / organic composite bio-ink loaded with cells A is 3 to 5 million / mL.

[0018] Preferably, the mass ratio of the network-like scaffold framework formed by the inorganic / organic composite bio-ink loaded with cells A to the scattered-point framework formed by the hydrogel bio-ink loaded with cells B is (1-3):1.

[0019] In another aspect, the present invention provides a method for preparing an inorganic / organic composite micropatterned multicellular scaffold, comprising:

[0020] (1) Using biological 3D printing technology to print inorganic / organic composite bio-ink loaded with cell A layer by layer, a three-dimensional network scaffold framework with a macroporous structure was prepared;

[0021] (2) filling the hydrogel bio-ink loaded with cell B into the pore structure of the three-dimensional network-like scaffold framework with a macroporous structure to obtain a composite three-dimensional scaffold;

[0022] (3) Cross-linking and curing the obtained composite three-dimensional scaffold to obtain the inorganic / organic composite micro-patterned multicellular scaffold; preferably, the cross-linking and curing temperature is 10 to 25° C., and the curing time is 30 to 60 seconds.

[0023] On the other hand, the present invention provides an application of an inorganic / organic composite micro-patterned multicellular scaffold in the preparation of skin tissue engineering materials, characterized in that the skin tissue engineering materials include hair regeneration materials, hair follicle reconstruction materials, and materials for treating androgenic alopecia.

[0024] Beneficial effects:

[0025] In the present disclosure, the inorganic / organic composite micro-patterned multicellular scaffold, in which two types of cells are distributed in a biomimetic pattern in three-dimensional space, simulates the hair follicle and vascular tissue structure in skin tissue, can promote hair follicle and blood vessel reconstruction, induce hair regeneration, and repair skin tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 (a) is the SEM image of magnesium silicate nanospheres (MS), (b) is the TEM image of magnesium silicate nanospheres (MS), (c) is the EDS image of the distribution of Mg, Si, and O elements in MS nanospheres, and (d) is the XRD pattern of MS nanospheres;

[0027] Figure 2 (a) is the SEM image of the interior of the GelMA / methylcellulose (GM) hybrid hydrogel matrix, (b) the rheological properties of the GM bio-ink matrix hydrogels with different MS concentrations at a shear rate range of 0.1-10s-1;

[0028] Figure 3 (a) shows a photo of 3D-printed micropatterned multicellular scaffolds (Co-GM, Co-2MS-GM, Co-4MS-GM, and Co-6MS-GM) with different MS contents. (b) shows the SEM image of the interior of the micropatterned multicellular scaffold, as well as the distribution of C, O, Mg, and Si elements and the merged EDS image.

[0029] Figure 4 (a) Fluorescence micrograph and (b) 3D image of cell distribution within the bio-3D-printed MS composite micropatterned multicellular scaffold, where the micropattern consists of grid-like distributed HUVECs cells (red) and dot-like distributed HHDPCs cells (green).

[0030] Figure 5 (a) Live-dead staining characterization (live cells: green, dead cells: red) and (b) cell survival rate statistics in the 3D-printed MS composite micropatterned multicellular scaffolds after 1, 7, 14, and 21 days of culture;

[0031] Figure 6 The cell morphological changes of HHDPCs in Co-GM, Co-2MS-GM, and Co-4MS-GM micropatterned multicellular scaffolds after 1 and 14 days of culture;

[0032] Figure 7 Expression of angiogenesis-related genes (VE-cad, KDR, HIF-1α, eNOs-1, and bFGF) and hair follicle development-related genes (c-Myc, PDGF-α, PDGF-β, and VEGF) in bio-3D-printed Co-GM, Co-2MS-GM, and Co-4MS-GM micropatterned multicellular scaffolds (*P < 0.05, **P < 0.01, ***P < 0.001, n = 4);

[0033] Figure 8 Comparison of the expression of angiogenesis-related genes and hair follicle formation-related genes in MS composite multicellular scaffolds (Co-2MS-GM) and MS composite single-cell scaffolds (EC-2MS-GM, DP-2MS-GM) (*P < 0.05, **P < 0.01, ***P < 0.001, n = 3);

[0034] Figure 9 In vivo transplantation experiments of 3D bioprinted micropatterned scaffolds, including: (a) skin wound photos and (b) statistical analysis of relative wound area on days 0, 8, 10, 12, and 14 after treatment with Blank, EC-2MS-GM (single-cell scaffold), Co-GM (multicellular scaffold), and Co-2MS-GM (multicellular scaffold) (**P < 0.01, ***P < 0.001, n = 5); (c) hair growth in the newborn skin of nude mice on day 30 (yellow arrow); H&E staining of skin samples from each group on day 14 (d) and day 30 (e) showing the appearance of new hair follicles in the Co-GM and Co-2MS-GM groups, with the Co-2MS-GM group having the largest number of regenerated hair follicles;

[0035] Figure 10Figure 3 Histological analysis of angiogenesis and hair follicle regeneration in nude mouse skin tissue. (a) CD31 immunofluorescence staining results showed that the Co-2MS-GM group had the most intensive angiogenesis in the skin tissue on day 14 (blue: cell nuclei, green: CD31). (b) Immunofluorescence staining results of the Co-2MS-GM group on day 30 showed strong positive expression of K5 (green) and AE13 (red) proteins, demonstrating that the MS inorganic / organic composite micropatterned multicellular scaffold had the best effect on hair follicle reconstruction.

[0036] Figure 11 Figure 3. In vivo skin repair and hair regeneration experiments in C57BL / 6 mice with androgenic alopecia (AGA). (a) Representative photographs of skin injury sites in the Blank, GM, and Co-2MS-GM groups after 0, 7, 15, 25, and 40 days of treatment. Statistics of relative wound area (c) and hair coverage (d) on days 0, 7, 15, 25, and 40, showing the prominent effect of Co-2MS-GM micropatterned multicellular scaffolds in promoting skin regeneration and hair regeneration. (b) SEM images and statistical analysis of hair diameters of newly formed hair in the Blank, GM, and Co-2MS-GM groups.

[0037] Figure 12 This is an H&E-stained photograph of mouse skin samples on day 25. Enlarging the black box clearly reveals the newly formed hair follicles in the Co-MS-GM group.

[0038] Figure 13 The immunohistochemical staining photos of Ki67 showed that the skin tissue of the Co-2MS-GM group was densely distributed with highly active hair follicles. DETAILED DESCRIPTION

[0039] The present invention is further described below through the following embodiments. It should be understood that the following embodiments are only used to illustrate the present invention, rather than to limit the present invention.

[0040] In the present disclosure, the inorganic / organic composite micropatterned multicellular scaffold can mimic the structure of hair follicles and vascular networks found in skin tissue, inducing hair and blood vessel regeneration during skin tissue repair. In an alternative embodiment, the bioactive inorganic material contained in the inorganic / organic composite micropatterned multicellular scaffold is a nanoparticle that can actively regulate cellular activity, preferably hollow magnesium silicate nanospheres.

[0041] In one embodiment of the present invention, the inorganic / organic composite micro-patterned multicellular scaffold utilizes a three-dimensional biomimetic micro-pattern composed of vascular endothelial cells and dermal papilla cells to simulate the punctate distribution of hair follicles and the reticular distribution of blood vessels in the skin.

[0042] The following is an exemplary description of the preparation method of the inorganic / organic composite micropatterned multicellular scaffold.

[0043] The hard template method was used to prepare magnesium silicate hollow nanospheres by hydrothermal treatment with SiO2 nanospheres as templates.

[0044] The synthesis method for SiO2 nanospheres is as follows: Weigh a certain amount of anhydrous ethanol and concentrated ammonia, mix them, and stir in a 30°C water bath. Weigh a small amount of ethyl orthosilicate (TES) and quickly pour it into the ethanol-ammonia mixture. Seal the cup and continue stirring for 1 hour. Centrifuge the mixture to obtain a white precipitate. This precipitate is ultrasonically washed with water and alcohol three times each, then placed in an oven until completely dry. A white powder, the SiO2 nanospheres, is obtained. The mass ratio of anhydrous ethanol to concentrated ammonia is 8:3, and the volume ratio of the mixture to TES is 25:1.

[0045] The preparation method for magnesium silicate hollow nanospheres is as follows: a certain amount of SiO2 nanosphere template is weighed, a certain amount of deionized water is added, and ultrasonic dispersion is performed for 30 minutes. A magnesium source and ammonium salt powders are weighed, and a solvent is added and stirred until the two powders are completely dissolved and mixed. The magnesium source is at least one of MgCl2, Mg(NO3)2, and MgSO4; the ammonium salt is at least one of NH4Cl, NH4NO3, and (NH4)2SO4; and the solvent is at least one of deionized water, pure water, or ultrapure water. A small amount of concentrated ammonia is drawn up using a syringe and slowly added dropwise to the magnesium salt-ammonium salt mixed solution. Stirring is continued for 30 minutes after the addition is complete. The ultrasonically treated SiO2 dispersion and anhydrous ethanol are quickly added and stirring is continued. Finally, the mixture is placed in a polytetrafluoroethylene hydrothermal reactor and hydrothermally reacted at 140°C for 12 hours. After the reaction is completed, the white precipitate at the bottom of the reactor is collected, ultrasonically washed with water and then with alcohol three times each, and then placed in a 60°C oven until completely dry, thereby obtaining magnesium silicate hollow nanospheres. The molar ratio of the Mg source to the ammonium salt weighed above is in the range of 1:10 to 1:20. The volume ratio of the magnesium salt-ammonium salt mixed solution, the SiO2 dispersion, and the anhydrous ethanol is 3:2:2.

[0046] Preparation of the inorganic / organic composite bio-ink loaded with HUVECs: A small amount of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator was weighed and dissolved in a certain amount of PBS buffer solution. Methacryl-modified gelatin (GelMA) was added to the solution and dissolved in a 65°C water bath in the dark to prepare the GelMA solution. A very small amount of methylcellulose (MC) was added to the solution and allowed to dissolve completely to obtain the GelMA-MC (GM) matrix material. After sterilization by filtration using a 0.22μm filter membrane, the solution was kept warm in a 37°C water bath until ready for use. A certain amount of magnesium silicate nanospheres was weighed and sterilized by UV crosslinking in a UV crosslinker for at least 1 hour. Sterile PBS buffer solution was then added to the magnesium silicate nanospheres, ultrasonically dispersed for 2 hours, and then thoroughly mixed with the GelMA-MC solution to obtain the magnesium silicate composite matrix hydrogel material. Human umbilical vein endothelial cells (HUVECs) cultured and expanded to the sixth passage were digested with trypsin, centrifuged, and mixed into the composite hydrogel matrix. The cells were then gently pipetted to evenly disperse them, resulting in an inorganic / organic composite bioink loaded with HUVECs.

[0047] Preparation of HHDPCs-loaded bio-ink: Dilute the prepared GelMA solution 2-fold with sterile PBS buffer. Use trypsin to digest human dermal papilla cells (HHDPCs) cultured and expanded to the third passage. After centrifugation, mix the cells into the diluted GelMA solution and use a pipette to gently disperse the cells. This creates the HHDPCs-loaded bio-ink.

[0048] Bio 3D printing process: This preparation process uses extrusion printing, as follows:

[0049] Framework preparation. The inorganic / organic composite bioink loaded with HUVECs was sealed in a metal cartridge and placed in a 4°C refrigerator for approximately 20 minutes to form a pregel. The cartridge was then loaded into the cooling printing channel of a 3D bioprinter, and printing was performed under sterile conditions. The cooling channel and printing platform were set to a temperature of 10°C, the extrusion printing pressure was approximately 30-70 kPa, and the printing needle was a 27G model with an inner diameter of approximately 250 μm. The printing program was set to a 90° interlayer rotation angle to produce a three-dimensional scaffold framework with a square macroporous structure.

[0050] Frame filling. After the HUVECs cell framework is printed, the bio-ink loaded with HHDPCs cells is fully filled into the framework's pores. Ultimately, the HUVECs framework and the HHDPCs filling form a single unit. The scaffold is then irradiated with blue light for approximately 1 minute to fully crosslink it, resulting in a 3D-printed micropatterned multicellular scaffold.

[0051] The scaffolds were transferred to a 12-well plate, and a mixed culture medium was prepared at a ratio of ECM:MSCM = 1:1. The mixed culture medium was added to each well and the plate was placed in a 37°C incubator for culture. The culture medium was changed every two days.

[0052] The following examples are further given to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention all fall within the scope of protection of the present invention. The specific process parameters and the like in the following examples are only examples within a suitable range, that is, those skilled in the art can make selections within a suitable range through the description herein, and are not limited to the specific numerical values ​​exemplified below.

[0053] Example 1

[0054] Preparation of hollow magnesium silicate nanospheres:

[0055] Weigh 64g of anhydrous ethanol and 24g of concentrated ammonia in a beaker, mix them, and stir in a 30°C waterbath for 10 minutes. Weigh 4.2mL of ethyl orthosilicate and quickly pour it into the ethanol-ammonia mixture. Seal the beaker and continue stirring for 1 hour. Centrifuge the mixture at 5000rpm / min for 5 minutes to obtain a white precipitate. Ultrasonicate the precipitate three times with water and three times with alcohol, then place it in a 60°C oven until completely dry. This yields a white powder, the SiO2 template.

[0056] Weigh 0.1g of SiO2 template, add 20mL of deionized water, and ultrasonically disperse for 30 minutes. Weigh 0.15g of magnesium chloride hexahydrate and 0.54g of ammonium chloride into the same beaker, add 30mL of deionized water, and stir until the powder is completely dissolved. Use a syringe to draw 1mL of concentrated ammonia water and slowly add it dropwise to the magnesium chloride-ammonium chloride mixture. Continue stirring for 30 minutes after the addition is complete. Quickly pour the ultrasonicated SiO2 dispersion and 25mL of anhydrous ethanol into the mixture and continue stirring for 1 hour. Finally, the mixture is placed in a 50mL polytetrafluoroethylene hydrothermal reactor and hydrothermally reacted at 140°C for 12 hours. After the reaction is completed, collect the white precipitate at the bottom of the reactor, ultrasonically rinse it with water and then with alcohol three times each, and place it in a 60°C oven until completely dry to obtain MS hollow nanospheres.

[0057] Figure 1 Figures a, b, and c show scanning electron microscopy, transmission electron microscopy, and EDS images of hollow magnesium silicate nanospheres prepared according to the above method, respectively. The MS has a hollow interior, a uniform shell thickness, and a rough exterior covered with nanosheets. Mg, Si, and O elements are evenly distributed throughout the MS nanospheres. Figure 1The d in the middle is the result of X-ray diffraction analysis, which proves that the magnesium silicate hollow nanospheres with regular morphology were prepared. The diameter of the nanospheres is about 400-500nm, which is Mg3Si4O with low crystallinity. 10 (OH)2 phase.

[0058] Example 2 Preparation of MS-GM Inorganic / Organic Composite Bio-ink:

[0059] Weigh 0.05g LAP photoinitiator and dissolve it in 10mL PBS buffer solution, add 1.2g GelMA thereto, and dissolve it in a 65℃ water bath in the dark. Wait until GelMA is completely dissolved to obtain a GelMA solution with a concentration of 12%. Add 0.01g methyl cellulose (MC) thereto, and after it is completely dissolved, obtain the GelMA-MC (GM) matrix material. After sterilization by filtering with a 0.22μm filter membrane, keep it warm in a 37℃ water bath for use. Weigh 0.0048g, 0.0096g, and 0.0144g MS powder (2%, 4%, and 6% of the mass of 2mL 12% GelMA) respectively, put them into a UV cross-linking instrument and sterilize them under UV light for more than 1h. 2 mL of sterile PBS buffer solution was then added to each MS, ultrasonically dispersed for 2 h, and then 2 mL of MS dispersion at different concentrations was thoroughly mixed with 2 mL of GelMA-MC solution to obtain four different MS-composite matrix hydrogel materials with different concentrations: GM, 2MS-GM, 4MS-GM, and 6MS-GM. HUVECs cultured and expanded to the sixth generation were digested with trypsin, centrifuged, and mixed into the four composite hydrogel matrices at a cell concentration of approximately 3-4 million / mL. The cells were slowly blown evenly using a pipette to evenly disperse them. The four bio-inks loaded with HUVECs after thorough mixing were designated EC-GM, EC-2MS-GM, EC-4MS-GM, and EC-6MS-GM.

[0060] Figure 2 Figure a shows that the MS-GM composite matrix hydrogel has a dense porous structure inside, which fully ensures the activity of cells in the gel. Figure 2 Figure b shows that the four bio-inks EC-GM, EC-2MS-GM, EC-4MS-GM, and EC-6MS-GM have shear-thinning properties and can be used for extrusion-type biological 3D printing.

[0061] Example 3 Preparation of Inorganic / Organic Composite Micropatterned Multicellular Scaffolds

[0062] Step (1): Preparation of MS-GM inorganic / organic composite bio-ink

[0063] Weigh 0.05g LAP photoinitiator and dissolve it in 10mL PBS buffer solution, add 1.2g GelMA thereto, and dissolve it in a 65℃ water bath in the dark. Wait until GelMA is completely dissolved to obtain a GelMA solution with a concentration of 12%. Add 0.01g methyl cellulose (MC) thereto, and after it is completely dissolved, obtain the GelMA-MC (GM) matrix material. After sterilization by filtering with a 0.22μm filter membrane, keep it warm in a 37℃ water bath for use. Weigh 0.0048g, 0.0096g, and 0.0144g MS powder (2%, 4%, and 6% of the mass of 2mL 12% GelMA) respectively, put them into a UV cross-linking instrument and sterilize them under UV light for more than 1h. After that, 2 mL of sterile PBS buffer solution was added to each MS, ultrasonically dispersed for 2 hours, and then 2 mL of MS dispersions of different concentrations were fully mixed with 2 mL of GelMA-MC solution to obtain GM, 2MS-GM, 4MS-GM, and 6MS-GM, four different concentrations of MS composite matrix hydrogel materials. HUVECs cultured and expanded to the sixth generation were digested with trypsin, centrifuged, and mixed into the four composite hydrogel matrices. The cell concentration was about 3-4 million / mL. A pipette was used to slowly blow the cells evenly to disperse them evenly. The four bio-inks loaded with HUVECs cells after thorough mixing were recorded as EC-GM, EC-2MS-GM, EC-4MS-GM, and EC-6MS-GM.

[0064] Step (2): Dilute the prepared 12% GelMA solution 2-fold with sterile PBS buffer to obtain a 6% GelMA solution. Use trypsin to digest the HHDPCs cultured and expanded to the third generation. After centrifugation, mix them into the 6% GelMA solution to a cell concentration of approximately 5 million / mL. Use a pipette to slowly blow the cells evenly to disperse them. This yields bio-ink 2 loaded with HHDPCs cells.

[0065] Step (3): Printing the MS-GM inorganic / organic composite micropatterned multicellular scaffold framework

[0066] Four types of bio-inks, EC-GM, EC-2MS-GM, EC-4MS-GM, and EC-6MS-GM, were sealed in metal barrels respectively and placed in a 4°C refrigerator for about 20 minutes to form a pre-gel. Each metal barrel was then loaded into the cooling printing channel of the biological 3D printer and printed separately under sterile conditions. The setting temperature of the cooling channel and the printing platform was 10°C, the air pressure of the extrusion printing used was about 20-40kPa, the printing needle model was 27G, and the inner diameter of the needle was about 250μm. The printing program was set to an inter-layer rotation angle of 90°, a square macroporous structure frame with a height of about 0.6mm, and a frame spacing of about 1.5mm (i.e., the side length of the square); a HUVECs cell frame with a porosity of about 40% was obtained;

[0067] Step (4): Frame filling

[0068] After printing the HUVEC cell framework, the pores of the framework were filled with bio-ink loaded with HHDPC cells. Ultimately, the HUVEC framework and the HHDPC-filled pores formed a single entity. The scaffold was irradiated with blue light for approximately 1 minute to fully crosslink it, resulting in an inorganic / organic composite micropatterned multicellular scaffold. The scaffold was transferred to a 12-well plate and a mixed culture medium was prepared at a ratio of ECM:MSCM = 1:1. 1 mL of the mixed culture medium was added to each well, and the scaffold was incubated at 37°C. The culture medium was changed every two days. Based on the four different MS content, the micropatterned multicellular scaffolds were designated as Co-GM (network scaffold framework to scattered filling mass ratio of 1.5:1), Co-2MS-GM (network scaffold framework to scattered filling mass ratio of 1.5:1), Co-4MS-GM (network scaffold framework to scattered filling mass ratio of 1.5:1), and Co-6MS-GM (network scaffold framework to scattered filling mass ratio of 1.5:1).

[0069] Figure 3 The appearance of four printed scaffolds is shown, with magnesium silicate nanospheres dispersed inside the scaffolds.

[0070] Example 4 Cell distribution of inorganic / organic composite micropatterned multicellular scaffolds

[0071] Step (1): Preparation of MS-GM inorganic / organic composite bio-ink

[0072] Weigh 0.05g LAP photoinitiator and dissolve it in 10mL PBS buffer solution, add 1.2g GelMA thereto, and dissolve it in a 65℃ water bath in the dark. Wait until GelMA is completely dissolved to obtain a GelMA solution with a concentration of 12%. Add 0.01g methyl cellulose (MC) thereto, and after it is completely dissolved, obtain the GelMA-MC (GM) matrix material, filter and sterilize it with a 0.22μm filter membrane, and keep it warm in a 37℃ water bath for use. Weigh 0.0048g MS powder (2% of the mass of 2mL 12% GelMA), place it in a UV cross-linking instrument and sterilize it under UV light for more than 1h. Then add 2mL of sterile PBS buffer solution to each MS, ultrasonically disperse it for 2h, and then fully mix 2mL MS dispersion with 2mL GelMA-MC solution to obtain 2MS-GM matrix hydrogel material. HUVECs cultured and expanded to the sixth generation were digested with trypsin, labeled with red fluorescence, and centrifuged before being mixed into the four composite hydrogel matrices at a concentration of approximately 3-4 million cells / mL. The cells were then gently pipetted to disperse them evenly. After thorough mixing, the HUVEC-loaded bioink, EC-2MS-GM, was obtained.

[0073] Step (2): Dilute the prepared 12% GelMA solution 2-fold with sterile PBS buffer to obtain a 6% GelMA solution. Use trypsin to digest the HHDPCs cultured and expanded to the third generation, label them with green fluorescence, centrifuge, and mix them into the 6% GelMA solution. The cell concentration is approximately 5 million / mL. Use a pipette to slowly blow the cells to evenly disperse them. This yields bio-ink 2 loaded with HHDPCs cells.

[0074] Step (3): Printing the MS-GM inorganic / organic composite micropatterned multicellular scaffold framework

[0075] The EC-2MS-GM bio-ink was sealed in a metal barrel and placed in a 4°C refrigerator for about 20 minutes to form a pre-gel. The metal barrel was then placed in the cooling printing channel of the biological 3D printer and printed under sterile conditions and in the dark. The setting temperature of the cooling channel and the printing platform was 10°C, the air pressure of the extrusion printing used was about 20-40kPa, the printing needle model was 27G, and the inner diameter of the needle was about 250μm. The printing program was set to an inter-layer rotation angle of 90°, a square macroporous structure frame with a height of about 0.6mm, and a frame spacing of about 1.5mm; a HUVECs cell frame with a porosity of about 40% was obtained;

[0076] Step (4): Frame filling

[0077] After the HUVECs cell frame is printed, the bio-ink loaded with HHDPCs cells is filled in the hole part of the frame. Finally, the HUVECs frame and the holes filled with HHDPCs form a whole. The scaffold is irradiated with blue light for about 1 minute to fully cross-link it, thereby obtaining a Co-2MS-GM inorganic / organic composite micro-patterned multicellular scaffold (the mass ratio of the network scaffold frame and the scattered point frame is 1.5:1). After the scaffold is transferred to a 12-well plate, a mixed culture medium is prepared in a ratio of ECM:MSCM=1:1. After adding 1 mL of mixed culture medium to each well, the scaffold is placed in a 37°C incubator for culture. The medium is changed every two days;

[0078] Step (5): Take photos and observe

[0079] After culturing the scaffolds in the dark for 1, 4, and 7 days, the distribution of the two cell types within the scaffolds was observed using confocal laser scanning microscopy. Lasers with wavelengths of 488 nm and 552 nm were used to excite the cells' green and red fluorescence, respectively. Planar images of the scaffolds were then converted to 3D images, yielding stereoscopic images of the cell distribution micropatterns.

[0080] Figure 4 A 3D image of the biomimetic cell distribution micropattern of a multicellular scaffold is shown. Red-fluorescent HUVEC bioink is arranged to form a vascular network "framework," while green-fluorescent HHDPC bioink is filled into the pores of the framework to mimic the punctate distribution of hair follicles. This micropattern structure remains stable during in vitro culturing of the scaffold.

[0081] Example 5 Cellular activity of inorganic / organic composite micropatterned multicellular scaffolds

[0082] Step (1): Preparation of MS-GM inorganic / organic composite bio-ink

[0083] Weigh 0.05g LAP photoinitiator and dissolve it in 10mL PBS buffer solution, add 1.2g GelMA thereto, and dissolve it in a 65℃ water bath in the dark. Wait until GelMA is completely dissolved to obtain a GelMA solution with a concentration of 12%. Add 0.01g methyl cellulose (MC) thereto, and after it is completely dissolved, obtain the GelMA-MC (GM) matrix material. After sterilization by filtering with a 0.22μm filter membrane, keep it warm in a 37℃ water bath for use. Weigh 0.0048g, 0.0096g, and 0.0144g MS powder (2%, 4%, and 6% of the mass of 2mL 12% GelMA) respectively, put them into a UV cross-linking instrument and sterilize them under UV light for more than 1h. After that, 2 mL of sterile PBS buffer solution was added to each MS, ultrasonically dispersed for 2 hours, and then 2 mL of MS dispersions of different concentrations were fully mixed with 2 mL of GelMA-MC solution to obtain GM, 2MS-GM, 4MS-GM, and 6MS-GM, four different concentrations of MS composite matrix hydrogel materials. HUVECs cultured and expanded to the sixth generation were digested with trypsin, centrifuged, and mixed into the four composite hydrogel matrices. The cell concentration was about 3-4 million / mL. A pipette was used to slowly blow the cells evenly to disperse them evenly. The four bio-inks loaded with HUVECs cells after thorough mixing were recorded as EC-GM, EC-2MS-GM, EC-4MS-GM, and EC-6MS-GM.

[0084] Step (2): Dilute the prepared 12% GelMA solution 2-fold with sterile PBS buffer to obtain a 6% GelMA solution. Use trypsin to digest the HHDPCs cultured and expanded to the third generation. After centrifugation, mix them into the 6% GelMA solution to a cell concentration of approximately 5 million / mL. Use a pipette to slowly blow the cells to evenly disperse them. This yields bio-ink 2 encapsulating HHDPCs cells.

[0085] Step (3): Printing the MS-GM inorganic / organic composite micropatterned multicellular scaffold framework

[0086] Four types of bio-inks, EC-GM, EC-2MS-GM, EC-4MS-GM, and EC-6MS-GM, were sealed in metal barrels respectively and placed in a refrigerator at 4°C for about 20 minutes to form a pre-gel. Each metal barrel was then loaded into the cooling printing channel of the biological 3D printer and printed separately under sterile conditions. The setting temperature of the cooling channel and the printing platform was 10°C, the air pressure of the extrusion printing used was about 20-40kPa, the printing needle model was 27G, and the inner diameter of the needle was about 250μm. The printing program was set to an inter-layer rotation angle of 90°, a square macroporous structure frame with a height of about 0.6mm, and a frame spacing of about 1.5mm; a HUVECs cell frame with a porosity of about 40% was obtained;

[0087] Step (4): Frame filling

[0088] After the HUVECs cell framework was printed, the bioink loaded with HHDPCs cells was filled into the pores of the framework. Ultimately, the HUVECs framework and the HHDPCs-filled pores formed a single entity. The scaffold was irradiated with blue light for approximately 1 minute to fully crosslink it, resulting in an inorganic / organic composite micropatterned multicellular scaffold. The scaffold was transferred to a 12-well plate and a mixed culture medium was prepared at a ratio of ECM:MSCM = 1:1. 1 mL of the mixed culture medium was added to each well and the scaffold was cultured in a 37°C incubator. The culture medium was changed every two days. Based on the four different MS contents, the micropatterned multicellular scaffolds were designated as Co-GM (network scaffold framework to scattered filling portion mass ratio of 1.5:1), Co-2MS-GM (network scaffold framework to scattered filling portion mass ratio of 1.5:1), Co-4MS-GM (network scaffold framework to scattered filling portion mass ratio of 1.5:1), and Co-6MS-GM (network scaffold framework to scattered filling portion mass ratio of 1.5:1).

[0089] Step (5): Cell live / dead staining and statistics

[0090] Live / dead fluorescence staining and photography were performed on the 1st, 7th, 14th, and 21st days of culture on the micropatterned scaffolds. A Calcein-AM / PI double staining kit was used to prepare a live / dead staining solution of AM:PI:ECM:MSCM=2:3:500:500. After removing the culture medium from the scaffold, the fluorescent staining solution was added to immerse the scaffold, and the scaffold was placed in a 37°C incubator and incubated for 20 minutes. Photography was taken using a fluorescence microscope. Cell viability was calculated by cell counting. The number of live cells and dead cells in the same area of ​​each group of fluorescence photos were calculated separately. Cell viability (%) = number of live cells / sum of the number of live cells and dead cells.

[0091] Figure 5The results show that the cell activity within the inorganic / organic composite micropatterned multicellular scaffolds. The Co-GM, Co-2MS-GM and Co-4MS-GM micropatterned multicellular scaffolds were able to maintain high survival of the two cell types within the scaffolds during a culture process of up to 21 days (green fluorescence). In contrast, the number of dead cells in the Co-6MS-GM micropatterned multicellular scaffold increased significantly (red fluorescence). Quantitative statistical results showed that the Co-GM and Co-2MS-GM scaffolds maintained a cell survival rate above 85% during the early culture process, and this rate rose to over 95% by day 21. The cell survival rate of the Co-4MS-GM scaffold was approximately 70% in the early culture stage, and by day 21 the survival rate had also increased to 85%. However, the cell survival rate within the Co-6MS-GM scaffold was always below 30%, indicating that the high content of MS had an adverse effect on cell survival.

[0092] Based on the above results, Co-GM, Co-2MS-GM and Co-4MS-GM are scaffolds with better biological activity.

[0093] Example 6 Cell Morphology of Inorganic / Organic Composite Micropatterned Multicellular Scaffold

[0094] Step (1): Preparation of MS-GM inorganic / organic composite bio-ink

[0095] Weigh 0.05 g of LAP photoinitiator and dissolve it in 10 mL of PBS buffer solution. Add 1.2 g of GelMA and dissolve it in a 65°C water bath protected from light. Wait for the GelMA to completely dissolve to obtain a 12% GelMA solution. Add 0.01 g of methylcellulose (MC) and wait for it to completely dissolve to obtain the GelMA-MC (GM) matrix material. After sterilization by filtration with a 0.22 μm filter membrane, keep it warm in a 37°C water bath until ready to use. Weigh 0.0048 g and 0.0096 g of MS powder (2% and 4% of the mass of 2 mL of 12% GelMA) respectively and place them in a UV crosslinker and sterilize them under UV light for at least 1 hour. After that, 2 mL of sterile PBS buffer solution was added to each MS, and ultrasonic dispersion was performed for 2 h. Then, 2 mL of MS dispersions of different concentrations were fully mixed with 2 mL of GelMA-MC solution to obtain three different concentrations of MS composite matrix hydrogel materials: GM, 2MS-GM, and 4MS-GM. HUVECs cultured and expanded to the sixth generation were digested with trypsin, centrifuged, and mixed into the four composite hydrogel matrices. The cell concentration was about 3-4 million / mL. The cells were slowly blown evenly with a pipette to disperse them evenly. The four bio-inks loaded with HUVECs after thorough mixing were recorded as EC-GM, EC-2MS-GM, and EC-4MS-GM.

[0096] Step (2): Dilute the prepared 12% GelMA solution 2-fold with sterile PBS buffer to obtain a 6% GelMA solution. Use trypsin to digest the HHDPCs cultured and expanded to the third generation. After centrifugation, mix them into the 6% GelMA solution. The cell concentration is approximately 5 million / mL. Use a pipette to slowly blow the cells to evenly disperse them. This yields bio-ink 2 loaded with HHDPCs cells.

[0097] Step (3): Printing the MS-GM inorganic / organic composite micropatterned multicellular scaffold framework

[0098] EC-GM, EC-2MS-GM, and EC-4MS-GM bio-inks were sealed in metal barrels respectively and placed in a 4°C refrigerator for about 20 minutes to form a pre-gel. Each metal barrel was then loaded into the cooling printing channel of the biological 3D printer and printed separately under sterile conditions. The setting temperature of the cooling channel and the printing platform was 10°C, the air pressure of the extrusion printing used was about 20-40kPa, the printing needle model was 27G, and the inner diameter of the needle was about 250μm. The printing program was set to an inter-layer rotation angle of 90°, a square macroporous structure frame with a height of about 0.6mm, and a frame spacing of about 1.5mm; a HUVECs cell frame with a porosity of about 40% was obtained;

[0099] Step (4): Frame filling

[0100] After the HUVECs cell framework is printed, the bio-ink loaded with HHDPCs cells is filled in the pores of the framework. Finally, the HUVECs framework and the pores filled with HHDPCs form a whole. The scaffold is irradiated with blue light for about 1 minute to fully cross-link it, thereby obtaining an inorganic / organic composite micro-patterned multicellular scaffold. After the scaffold is transferred to a 12-well plate, a mixed culture medium is prepared in a ratio of ECM:MSCM = 1:1. After adding 1 mL of mixed culture medium to each well, the scaffold is placed in a 37°C incubator for culture. The medium is changed every two days. According to the four different contents of MS, the micro-patterned multicellular scaffolds are respectively recorded as Co-GM (the mass ratio of the network scaffold framework and the scattered filling part is 1.5:1), Co-2MS-GM (the mass ratio of the network scaffold framework and the scattered filling part is 1.5:1), and Co-4MS-GM (the mass ratio of the network scaffold framework and the scattered filling part is 1.5:1);

[0101] Step (5): Staining of cell nuclei / skeleton inside the scaffold

[0102] After 1 and 14 days of in vitro culture, the 3D-printed bioscaffolds were fixed in 4% paraformaldehyde for at least 30 minutes, washed with PBS, and placed on a shaker. The cell nuclei and cytoskeleton were then stained. AlexFluor 647-conjugated phalloidin fluorescent dye was added and stained for 1 hour at room temperature in the dark. After washing with PBS buffer, DAPI fluorescent dye was added and the cells were stained for 10 minutes in the dark on a shaker. After staining, fluorescence micrographs of the HHDPC nuclei and cytoskeleton were observed using confocal laser scanning microscopy using excitation wavelengths of 405 nm and 647 nm.

[0103] Figure 6 It was shown that hair follicle stem cells migrated and aggregated into three-dimensional spheres during the culture process of 1 to 14 days. The sphere formation was able to partially simulate the structure of the dermal hair papilla, providing a basis for hair follicle formation, and proving the high activity of HHDPCs cells in the micropatterned multicellular scaffold.

[0104] Example 7 Angiogenic and hair follicle-forming gene expression of inorganic / organic composite micropatterned multicellular scaffolds Step (1): Preparation of MS-GM inorganic / organic composite bio-ink: Weigh 0.05 g of LAP photoinitiator and dissolve it in 10 mL of PBS buffer solution. Add 1.2 g of GelMA and dissolve it in a 65°C water bath in the dark. Wait for GelMA to completely dissolve to obtain a 12% GelMA solution. Add 0.01 g of methylcellulose (MC) and wait for it to completely dissolve to obtain a GelMA-MC (GM) matrix material. After sterilization by filtration with a 0.22 μm filter membrane, keep it warm in a 37°C water bath for use. Weigh 0.0048 g and 0.0096 g of MS powder (2% and 4% of the mass of 2 mL of 12% GelMA) respectively and place them in a UV crosslinker and sterilize them under UV light for more than 1 hour. After that, 2 mL of sterile PBS buffer solution was added to each MS, and ultrasonic dispersion was performed for 2 hours. Then, 2 mL of MS dispersions of different concentrations were fully mixed with 2 mL of GelMA-MC solution to obtain three different concentrations of MS composite matrix hydrogel materials: GM, 2MS-GM, and 4MS-GM. HUVECs cultured and expanded to the sixth generation were digested with trypsin, centrifuged, and mixed into the four composite hydrogel matrices. The cell concentration was about 3-4 million / mL. A pipette was used to slowly blow the cells evenly to disperse them. The four bio-inks loaded with HUVECs cells after thorough mixing were recorded as EC-GM, EC-2MS-GM, and EC-4MS-GM.

[0105] Step (2): Dilute the prepared 12% GelMA solution 2-fold with sterile PBS buffer to obtain a 6% GelMA solution. Use trypsin to digest the HHDPCs cultured and expanded to the third generation. After centrifugation, mix them into the 6% GelMA solution to a cell concentration of approximately 5 million / mL. Use a pipette to slowly blow the cells evenly to disperse them. This yields bio-ink 2 loaded with HHDPCs cells.

[0106] Step (3): Printing MS-GM inorganic / organic composite micro-patterned multicellular scaffold framework: EC-GM, EC-2MS-GM, and EC-4MS-GM three kinds of biological inks were sealed in metal barrels respectively and placed in a 4°C refrigerator for about 20 minutes to form a pre-gel. After that, each metal barrel was loaded into the cooling printing channel of the biological 3D printer and printed separately under sterile conditions. The setting temperature of the cooling channel and the printing platform was 10°C, the air pressure of the extrusion printing used was about 20-40kPa, the printing needle model was 27G, and the inner diameter of the needle was about 250μm. The printing program was set to an inter-layer rotation angle of 90°, a square macroporous structure frame with a height of about 0.6mm, and a frame spacing of about 1.5mm; a HUVECs cell frame with a porosity of about 40% was obtained;

[0107] Step (4): Frame filling: After the HUVECs cell frame is printed, the bio-ink loaded with HHDPCs cells is filled in the pores of the frame. Finally, the HUVECs frame and the pores filled with HHDPCs form a whole. The scaffold is irradiated with blue light for about 1 minute to fully cross-link it, thereby obtaining an inorganic / organic composite micro-patterned multicellular scaffold. After the scaffold is transferred to a 12-well plate, a mixed culture medium is prepared in a ratio of ECM:MSCM = 1:1. After adding 1 mL of mixed culture medium to each well, the scaffold is placed in a 37°C incubator for culture. The medium is changed every two days. According to the four different contents of MS, the micro-patterned multicellular scaffolds are respectively recorded as Co-GM (the mass ratio of the network scaffold frame and the scattered filling part is 1.5:1), Co-2MS-GM (the mass ratio of the network scaffold frame and the scattered filling part is 1.5:1), and Co-4MS-GM (the mass ratio of the network scaffold frame and the scattered filling part is 1.5:1);

[0108] Step (5): Detection of the expression levels of genes related to angiogenesis and hair follicle differentiation within the scaffolds: After 7 days of culture, the Co-GM, Co-2MS-GM, and Co-4MS-GM scaffolds were lysed for 1.5-2 hours, centrifuged, and the precipitate was collected. The cells within the scaffolds were extracted. 1 ml of Trizol reagent was added to the cells to lyse the cells and extract the intracellular RNA. The RNA was reverse transcribed using the PrimeScript 1stStrand kit to obtain cDNA. Angiogenesis-related genes and hair follicle differentiation-related genes in each group of scaffolds were detected using an RT-qPCR instrument.

[0109] Figure 7 The results showed that the expression of five angiogenic genes (VE-cad, KDR, HIF-1α, eNOs-1, and bFGF) and four hair follicle differentiation-related genes (c-Myc, PDGF-α, PDGF-β, and VEGF) in the Co-2MS-GM micropatterned multicellular scaffolds were significantly increased. Therefore, the Co-2MS-GM inorganic / organic composite micropatterned multicellular scaffold has both high angiogenic and hair follicle-forming activity.

[0110] Example 8 Comparison of the biological activities of inorganic / organic composite micropatterned multicellular scaffolds and single-cell scaffolds Step (1): Preparation of MS-GM inorganic / organic composite bio-ink: Weigh 0.05g of LAP photoinitiator and dissolve it in 10mL of PBS buffer solution, add 1.2g of GelMA thereto, and dissolve it in a water bath at 65°C in the dark. Wait for GelMA to completely dissolve to obtain a GelMA solution with a concentration of 12%. Add 0.01g of methyl cellulose (MC) thereto, and wait for it to completely dissolve to obtain the GelMA-MC (GM) matrix material. After sterilization by filtering with a 0.22μm filter membrane, keep it warm in a water bath at 37°C for use. Weigh 0.0048g of MS powder (2% of the mass of 2mL 12% GelMA) and place it in a UV crosslinker and sterilize it under UV light for more than 1h. 2 mL of sterile PBS buffer solution was then added to each MS, ultrasonically dispersed for 2 hours, and then 2 mL of MS dispersion was thoroughly mixed with 2 mL of GelMA-MC solution to obtain a 2MS-GM matrix hydrogel material. HUVECs cultured and expanded to the sixth generation were digested with trypsin, labeled with red fluorescence, and centrifuged before being mixed into the four composite hydrogel matrices at a cell concentration of approximately 3-4 million / mL. The cells were gently blown evenly with a pipette to disperse them uniformly. After thorough mixing, the HUVEC-loaded bioink EC-2MS-GM was obtained.

[0111] Step (2): Dilute the prepared 12% GelMA solution 2-fold with sterile PBS buffer to obtain a 6% GelMA solution. Use trypsin to digest the HHDPCs cultured and expanded to the third generation, label them with green fluorescence, centrifuge, and mix them into the 6% GelMA solution. The cell concentration is approximately 5 million / mL. Use a pipette to slowly blow the cells to evenly disperse them. This yields bio-ink 2 loaded with HHDPCs cells.

[0112] Step (3): Printing MS-GM inorganic / organic composite micro-patterned multicellular scaffold framework: seal the EC-2MS-GM bio-ink into a metal barrel and place it in a 4°C refrigerator for about 20 minutes to form a pre-gel. Then, load the metal barrel into the cooling printing channel of the biological 3D printer and print under sterile conditions and away from light. The setting temperature of the cooling channel and the printing platform is 10°C, the air pressure of the extrusion printing used is about 20-40kPa, the printing needle model is 27G, and the inner diameter of the needle is about 250μm. The printing program is set to an inter-layer rotation angle of 90°, a square macroporous structure frame with a height of about 0.6mm, and a frame spacing of about 1.5mm; a HUVECs cell frame with a porosity of about 40% is obtained;

[0113] Step (4): Frame filling: After the HUVECs cell frame is printed, the bio-ink loaded with HHDPCs cells is filled in the hole part of the frame. Finally, the HUVECs frame and the holes filled with HHDPCs form a whole. Use blue light to irradiate the scaffold for about 1 minute to fully cross-link it, and then obtain the Co-2MS-GM inorganic / organic composite micro-patterned multicellular scaffold (the mass ratio of the network scaffold frame and the scattered point frame is 1.5:1). After the scaffold is transferred to a 12-well plate, a mixed culture medium is prepared with an ECM:MSCM = 1:1 ratio. After adding 1 mL of mixed culture medium to each well, the scaffold is placed in a 37°C incubator for culture. The medium is changed every two days;

[0114] Step (5): Preparation of single-cell scaffolds: Using the same method, inorganic / organic composite single-cell scaffolds EC-2MS-GM and DP-2MS-GM containing only HUVECs and HHDPCs were prepared, respectively. The distribution of MS and cells in the two single-cell scaffolds was consistent with the corresponding material and cell distribution in the micropatterned multicellular scaffold.

[0115] Step (6): Gene expression in multicellular and single-cell scaffolds: After 7 days of culture, the Co-2MS-GM, EC-2MS-GM, and DP-2MS-GM scaffolds were lysed for 1.5-2 hours, centrifuged, and the precipitate was collected. The cells within the scaffolds were then extracted. 1 ml of Trizol reagent was added to the cells to lyse them and extract intracellular RNA. The RNA was reverse transcribed using the PrimeScript 1st Strand kit to generate cDNA. Angiogenesis-related genes and hair follicle differentiation-related genes in each group of scaffolds were detected using an RT-qPCR instrument.

[0116] Figure 8 The expression levels of four angiogenic genes, VEGF, HIF-1α, KDR, and VE-cad, were significantly higher in the Co-2MS-GM scaffold co-culture system than in the EC-2MS-GM monoculture system, demonstrating that the addition of HHDPCs can further enhance the angiogenic capacity of the three-dimensional system. Furthermore, compared to the DP-2MS-GM scaffold monoculture system, the presence of HUVECs in the co-culture system positively promoted the expression of hair follicle-related genes, c-Myc, PDGF-β, and VEGF. These results demonstrate the superiority of the constructed Co-2MS-GM micropatterned multicellular co-culture system.

[0117] Example 9 Tissue repair effect of inorganic / organic composite micropatterned multicellular scaffold on nude mouse skin damage Step (1): Preparation of MS-GM inorganic / organic composite bio-ink: Weigh 0.05g LAP photoinitiator and dissolve it in 10mL PBS buffer solution, add 1.2g GelMA thereto, and dissolve it in a water bath at 65℃ away from light. Wait until GelMA is completely dissolved to obtain a GelMA solution with a concentration of 12%. Add 0.01g methyl cellulose (MC) thereto, and wait until it is completely dissolved to obtain a GelMA-MC (GM) matrix material. After sterilization by filtration with a 0.22μm filter membrane, keep it warm in a water bath at 37℃ for use. Weigh 0.0048g MS powder (2% of the mass of 2mL 12% GelMA) and place it in a UV crosslinker and sterilize it under UV light for more than 1h. 2 mL of sterile PBS buffer solution was then added to each MS, ultrasonically dispersed for 2 hours, and then 2 mL of MS dispersion was thoroughly mixed with 2 mL of GelMA-MC solution to obtain a 2MS-GM matrix hydrogel material. HUVECs cultured and expanded to the sixth generation were digested with trypsin, labeled with red fluorescence, and centrifuged before being mixed into the four composite hydrogel matrices at a cell concentration of approximately 3-4 million / mL. The cells were gently blown evenly with a pipette to disperse them uniformly. After thorough mixing, the HUVEC-loaded bioink EC-2MS-GM was obtained.

[0118] Step (2): Dilute the prepared 12% GelMA solution 2-fold with sterile PBS buffer to obtain a 6% GelMA solution. Use trypsin to digest the HHDPCs cultured and expanded to the third generation, label them with green fluorescence, centrifuge, and mix them into the 6% GelMA solution. The cell concentration is approximately 5 million / mL. Use a pipette to slowly blow the cells to evenly disperse them. This yields bio-ink 2 loaded with HHDPCs cells.

[0119] Step (3): Printing MS-GM inorganic / organic composite micro-patterned multicellular scaffold framework: seal the EC-2MS-GM bio-ink into a metal barrel and place it in a 4°C refrigerator for about 20 minutes to form a pre-gel. Then, load the metal barrel into the cooling printing channel of the biological 3D printer and print under sterile conditions and away from light. The setting temperature of the cooling channel and the printing platform is 10°C, the air pressure of the extrusion printing used is about 20-40kPa, the printing needle model is 27G, and the inner diameter of the needle is about 250μm. The printing program is set to an inter-layer rotation angle of 90°, a circular macroporous structure frame with a height of about 0.8mm, a circle radius of 5mm, and a frame spacing of about 1.5mm; a HUVECs cell framework with a porosity of about 40% is obtained;

[0120] Step (4): Frame filling: After the HUVECs cell frame is printed, the bio-ink loaded with HHDPCs cells is filled in the hole part of the frame. Finally, the HUVECs frame and the holes filled with HHDPCs form a whole. Use blue light to irradiate the scaffold for about 1 minute to fully cross-link it, and then obtain the Co-2MS-GM inorganic / organic composite micro-patterned multicellular scaffold. After the scaffold is transferred to a 12-well plate, a mixed culture medium is prepared with an ECM:MSCM = 1:1 ratio. After adding 1 mL of mixed culture medium to each well, the scaffold is placed in a 37°C incubator for culture. The medium is changed every two days;

[0121] Step (5): The same method was used to prepare the single-cell scaffold EC-2MS-GM containing only HUVECs and the micropatterned multicellular scaffold Co-GM without magnesium silicate nanospheres;

[0122] Step (6): Establishment and treatment of full-thickness skin injury model in nude mice: Male 8-week-old nude mice (SPF clean grade) were selected for full-thickness skin defect repair experiments. The nude mice were randomly divided into 4 groups: blank group (no scaffold), EC-2MS-GM group, Co-GM group and Co-2MS-GM group. All scaffolds were cultured in vitro for 3 days before being used for wound transplantation. Before surgery, the nude mice were anesthetized by intraperitoneal injection of sodium pentobarbital. After the skin of the nude mice was disinfected, a circular full-thickness skin defect wound with a diameter of about 10 mm was created on its back with surgical scissors. After the scaffold was transplanted to the skin defect site, the scaffold was fixed and bandaged with sutures, sterile gauze and medical dressings. The nude mice were raised in groups in an SPF environment;

[0123] Step (7): The backs of the nude mice were photographed on days 0, 8, 10, 12, and 14. The actual wound area was measured, and the relative wound area of ​​each group at each time point was calculated. Hair growth on the backs of the nude mice was recorded on day 32. Skin tissue samples from the wounds were collected on days 14 and 32 and analyzed by histological staining.

[0124] Figure 9 The wound healing status of each group within 14 days is shown. Based on wound photographs and relative wound area statistics, the Co-2MS-GM group had the fastest skin wound healing rate, followed by the EC-2MS-GM and Co-GM groups. This demonstrates that the MS-composite micropatterned multicellular scaffold effectively promotes skin wound healing. After 32 days, hair growth was observed on the dorsal skin surface of mice in the Co-GM and Co-2MS-GM groups. Histological staining results show that the wound healing rate over the first 14 days was highest in the Co-2MS-GM group, followed by the Co-GM group, the EC-2MS-GM group, and the Blank group. By day 14, dermal reconstruction and re-epithelialization were complete in the Co-2MS-GM group, while unhealed epidermis and scars were still present in the skin samples of the other groups (black arrows). By day 30, the skin tissue of the Co-2MS-GM group was densely distributed with hair follicles, while the number of hair follicles in the Co-GM scaffold was slightly smaller, and no hair follicle structures were observed in the other groups.

[0125] Figure 10The results of immunofluorescence staining of CD31 (endothelial cell junction marker), K5 (outer root sheath marker) and AE13 (inner root sheath marker) proteins in skin tissue are shown to characterize the formation of blood vessels and hair follicles during skin regeneration. Comparing the EC-2MS-GM group and the Co-GM group, the MS and HHDPC contained in the Co-2MS-GM can significantly promote angiogenesis. In addition, functional hair follicles were detected in the Co-2MS-GM group at 30 days. Compared with the Co-GM group, the newly formed hair follicles in the Co-2MS-GM group were more densely distributed, some of which grew in an elongated morphology to the subdermis, and the AE13 positive reaction was strong. Therefore, the Co-2MS-GM inorganic / organic composite micropatterned multicellular scaffold has high biological activity for both hair regeneration and vascular reconstruction.

[0126] Example 10 Effect of inorganic / organic composite micropatterned multicellular scaffold on hair regeneration in mice with sex hormone-induced alopecia: Step (1): Preparation of MS-GM inorganic / organic composite bio-ink: Weigh 0.05g of LAP photoinitiator and dissolve it in 10mL of PBS buffer solution, add 1.2g of GelMA thereto, and dissolve it in a water bath at 65°C in the dark. Wait for GelMA to completely dissolve to obtain a GelMA solution with a concentration of 12%. Add 0.01g of methyl cellulose (MC) thereto, and wait for it to completely dissolve to obtain a GelMA-MC (GM) matrix material. After sterilization by filtration with a 0.22μm filter membrane, keep it warm in a water bath at 37°C for use. Weigh 0.0048g of MS powder (2% of the mass of 2mL 12% GelMA) and place it in a UV crosslinker and sterilize it under UV light for more than 1h. 2 mL of sterile PBS buffer solution was then added to each MS, ultrasonically dispersed for 2 hours, and then 2 mL of MS dispersion was thoroughly mixed with 2 mL of GelMA-MC solution to obtain a 2MS-GM matrix hydrogel material. HUVECs cultured and expanded to the sixth generation were digested with trypsin, labeled with red fluorescence, and centrifuged before being mixed into the four composite hydrogel matrices at a cell concentration of approximately 3-4 million / mL. The cells were gently blown evenly with a pipette to disperse them uniformly. After thorough mixing, the HUVEC-loaded bioink EC-2MS-GM was obtained.

[0127] Step (2): Dilute the prepared 12% GelMA solution 2-fold with sterile PBS buffer to obtain a 6% GelMA solution. Use trypsin to digest the HHDPCs cultured and expanded to the third generation, label them with green fluorescence, centrifuge, and mix them into the 6% GelMA solution. The cell concentration is approximately 5 million / mL. Use a pipette to slowly blow the cells to evenly disperse them. This yields bio-ink 2 loaded with HHDPCs cells.

[0128] Step (3): Printing MS-GM inorganic / organic composite micro-patterned multicellular scaffold framework: seal the EC-2MS-GM bio-ink into a metal barrel and place it in a 4°C refrigerator for about 20 minutes to form a pre-gel. Then, load the metal barrel into the cooling printing channel of the biological 3D printer and print under sterile conditions and away from light. The setting temperature of the cooling channel and the printing platform is 10°C, the air pressure of the extrusion printing used is about 20-40kPa, the printing needle model is 27G, and the inner diameter of the needle is about 250μm. The printing program is set to an inter-layer rotation angle of 90°, a circular macroporous structure frame with a height of about 0.8mm, a circle radius of 5mm, and a frame spacing of about 1.5mm; a HUVECs cell framework with a porosity of about 40% is obtained;

[0129] Step (4): Frame filling: After the HUVECs cell frame is printed, the bio-ink loaded with HHDPCs cells is filled in the hole part of the frame. Finally, the HUVECs frame and the holes filled with HHDPCs form a whole. Use blue light to irradiate the scaffold for about 1 minute to fully cross-link it, and then obtain the Co-2MS-GM inorganic / organic composite micro-patterned multicellular scaffold. After the scaffold is transferred to a 12-well plate, a mixed culture medium is prepared with an ECM:MSCM = 1:1 ratio. After adding 1 mL of mixed culture medium to each well, the scaffold is placed in a 37°C incubator for culture. The medium is changed every two days;

[0130] Step (5): using the same method to prepare a hydrogel scaffold GM without cells and magnesium silicate nanospheres;

[0131] Step (6): Establishment and treatment of androgenic alopecia (AGA) mouse model: 8-week-old male C57BL / 6 mice were used to establish an androgenic alopecia (AGA) model. C57BL / 6 mice were randomly divided into three groups: blank group (no scaffold), GM group and Co-2MS-GM group. Mice were subcutaneously injected with testosterone solution (5 mg / mL) daily. All scaffolds were cultured in vitro for 3 days before being used for wound transplantation. On the 7th day after injection, AGA mice were anesthetized by intraperitoneal injection of sodium pentobarbital. After the mouse skin was disinfected, a circular full-thickness skin defect wound with a diameter of about 10 mm was created on its back with surgical scissors. After the scaffold was transplanted into the skin defect site, the scaffold was fixed and bandaged with sutures, sterile gauze and medical dressings. Testosterone solution was then injected daily for three consecutive weeks. AGA mice were raised in groups in an SPF environment;

[0132] Step (7): The backs of the mice were photographed on days 0, 7, 15, 25, and 40. The actual wound area was measured, and the relative wound area of ​​each group at each time point was calculated. Hair was collected on day 40 for scanning electron microscopy observation, and skin tissue samples were collected on days 25 and 40 for histological staining and analysis of the wound skin tissue samples.

[0133] AGA is a chronic disease characterized by hair loss and degeneration of hair follicles. Figure 11 The results show wound healing and hair growth on the backs of AGA mice over a 40-day period. Of the three groups, the Co-2MS-GM group showed the fastest wound healing, with melanin pigmentation appearing by day 15 and dense hair regeneration by day 40. In contrast, the other two groups showed minimal new hair growth. Statistical analysis of the diameter of newly regenerated hair revealed that the Co-2MS-GM group had intact scales and thicker hair diameter than the other two groups, indicating higher hair quality.

[0134] Figure 12 and Figure 13 Shown are histological staining results of AGA mouse skin tissue. HE staining results demonstrate the highest number of newly formed hair follicles in the Co-2MS-GM group, while the blank and GM groups have fewer hair follicles. Immunohistochemical staining for Ki67 (a cell proliferation marker) reveals a dense distribution of highly active hair follicles in the Co-2MS-GM group, demonstrating the remarkable effectiveness of the Co-2MS-GM micropatterned multicellular scaffold in inducing hair follicle development.

Claims

1. An inorganic / organic composite micro-patterned multicellular scaffold, characterized in that: include: A three-dimensional network-like scaffold framework is formed by arranging inorganic / organic composite bio-ink loaded with cell A, and a scattered point framework is formed by arranging hydrogel bio-ink loaded with cell B filled in the pore structure of the three-dimensional network-like scaffold framework; the inorganic / organic composite bio-ink loaded with cell A comprises cell A, a bioactive inorganic material and a first hydrogel matrix, the cell A is a vascular endothelial cell, the bioactive inorganic material is a magnesium silicate nanohollow sphere, the first hydrogel matrix comprises at least one of methacrylated gelatin, methacrylated hyaluronic acid, collagen, and methylcellulose, and the mass of the bioactive inorganic material does not exceed 8wt% of the mass of the first hydrogel matrix; the hydrogel bio-ink loaded with cell B comprises cell B and a second hydrogel matrix, the cell B is a dermal papilla cell, and the second hydrogel matrix comprises at least one of methacrylated gelatin, methacrylated hyaluronic acid, collagen, and methylcellulose; the three-dimensional network-like scaffold framework and the scattered point framework form a biomimetic pattern.

2. The inorganic / organic composite micro-patterned multicellular scaffold according to claim 1, characterized in that: The three-dimensional network-shaped scaffold framework formed by the arrangement of the inorganic / organic composite biological ink loaded with cells A has a porosity of 30-50%, and a diameter of the pore structure is 800 μm-2 mm.

3. The micropatterned multicellular scaffold according to claim 1, wherein: The cell A is one of human umbilical vein endothelial cells and human dermal microvascular endothelial cells; the particle size of the magnesium silicate hollow nanospheres is 400-600 nm.

4. The micropatterned multicellular scaffold according to claim 1, wherein The cell B is one of a human dermal papilla cell and a human hair follicle stem cell.

5. The micropatterned multicellular scaffold according to claim 1, characterized in that The loading amount of cells B in the hydrogel bio-ink loaded with cells B is 3 million / mL to 5 million / mL.

6. The micropatterned multicellular scaffold according to claim 1, characterized in that The first hydrogel matrix is ​​a uniform mixture of methacrylated gelatin and methylcellulose.

7. The micropatterned multicellular scaffold according to claim 6, characterized in that: The first hydrogel matrix is ​​a uniform mixture of methacrylated gelatin and methyl cellulose, wherein the mass ratio of methacrylated gelatin to methyl cellulose is (100-150):

1.

8. The micropatterned multicellular scaffold according to claim 1, wherein The second hydrogel matrix is ​​methacrylated gelatin.

9. The micropatterned multicellular scaffold according to claim 1, wherein The mass of the bioactive inorganic material is 0.1 to 6 wt % of the mass of the first hydrogel matrix; the loading amount of cells A in the inorganic / organic composite bio-ink loaded with cells A is 3 million / mL to 5 million / mL.

10. The inorganic / organic composite micro-patterned multicellular scaffold according to claim 1, characterized in that: The mass ratio of the network-like scaffold frame formed by the inorganic / organic composite bio-ink loaded with cells A to the scattered-point frame formed by the hydrogel bio-ink loaded with cells B is (1-3):

1.

11. A method for preparing an inorganic / organic composite micro-patterned multicellular scaffold according to any one of claims 1 to 10, characterized in that: include: (1) Using biological 3D printing technology to print inorganic / organic composite bio-ink loaded with cell A layer by layer, a three-dimensional network scaffold framework with a macroporous structure was prepared; (2) filling the cell B-loaded hydrogel bio-ink into the pore structure of the three-dimensional network scaffold framework with a macroporous structure to obtain a composite three-dimensional scaffold; (3) Cross-linking and curing the obtained composite three-dimensional scaffold to obtain the inorganic / organic composite micro-patterned multicellular scaffold; preferably, the cross-linking and curing temperature is 10 to 25° C., and the curing time is 30 to 60 seconds.

12. Use of the inorganic / organic composite micro-patterned multicellular scaffold according to any one of claims 1 to 10 in preparing skin tissue engineering materials, characterized in that: The skin tissue engineering materials include hair regeneration materials, hair follicle reconstruction materials, and materials for treating androgenic alopecia.

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