A method for constructing ordered skin-like tissue with magnetic field assistance

Abstract

This invention belongs to the field of skin-like tissue preparation technology, and relates to a method for constructing ordered skin-like tissue with magnetic field assistance. The method involves directional cryo-casting to prepare a layered ordered hydrogel, providing skin-like seed cells, and culturing and expanding these cells in large quantities using a complete culture medium to obtain skin-like cells. The cultured skin-like cells are then co-cultured with iron oxide nanoparticles to prepare magnetized skin-like cells. These magnetized skin-like cells are then co-cultured with the layered ordered hydrogel, and magnetic fields are applied to both sides to induce the magnetized skin-like cells to enter the layered ordered hydrogel, resulting in a hydrogel scaffold. After washing the obtained hydrogel scaffold, it is transferred to a complete culture medium, and keratinocytes are seeded on the surface of the hydrogel scaffold and cultured to prepare an ordered skin-like tissue. This invention can effectively regulate the migration and arrangement of fibroblasts, providing a physical basis for the arrangement of the extracellular matrix, especially collagen.

Description

Technical Field

[0001] This invention belongs to the field of skin-like tissue preparation technology, and in particular relates to a method for constructing ordered skin-like tissue with magnetic field assistance. Background Technology

[0002] Difficult wound healing is a common problem in clinical treatment. Under normal circumstances, skin tissue has a certain capacity for self-repair and reconstruction, but when the wound area exceeds its repair threshold, the skin tissue cannot fully heal, forming a chronic wound. The persistent inflammatory response and disordered fibroblast arrangement in chronic wounds are one of the important mechanisms leading to scar formation. To achieve high-quality repair results, skin grafting is often considered the preferred method for treating chronic wounds. Statistics show that approximately 15% to 25% of diabetic patients will experience chronic wounds of varying degrees throughout their lives. However, skin grafting also carries some risks, such as secondary trauma, limited transplant sources, immune rejection, and the risk of disease transmission.

[0003] Skin tissue engineering is an emerging approach to address the problems inherent in traditional skin grafts. It utilizes combinations of biomaterials, cells, and biological factors to mimic the structure and function of natural skin tissue, thereby promoting regeneration and healing of damaged sites. The primary goal of skin tissue engineering is to develop alternative treatments to traditional skin grafts, addressing issues such as limited transplant sources, immune rejection, and secondary trauma. By combining biomaterials and cells, it creates biocompatible and bioactive artificial skin tissue, providing new avenues for chronic wound repair.

[0004] Skin-like structures are artificial skin tissues cultured in vitro in three dimensions, capable of mimicking the structure and function of natural skin. With the assistance of external materials, skin-like structures can be formed through a series of cellular activities regulated by mammalian pluripotent stem cells or adult tissues, including proliferation, differentiation, and self-organization migration. Compared to traditional cell loading methods, skin-like structures possess a complex structure similar to natural skin tissue, including multi-layered microstructures and macroscopic morphology, thereby improving the functionality and durability of the repaired area.

[0005] Current skin-like tissue reconstructions primarily focus on replicating skin cell components. This is mainly achieved by loading seed cells such as keratinocytes and fibroblasts into scaffold materials like hydrogels to simulate the bioactivity of skin tissue. Although these constructs possess some healing-promoting capabilities, they typically neglect the highly ordered structure unique to natural skin tissue, resulting in poor healing-promoting abilities and skin-like properties. Summary of the Invention

[0006] Because existing technologies for preparing skin-like tissues neglect the highly ordered structure unique to natural skin tissue, their healing ability and skin-like properties are poor. To address this problem, this invention provides a method for constructing ordered skin-like tissues with magnetic field assistance.

[0007] This invention utilizes an ordered hydrogel structure combined with a magnetic field to guide fibroblast migration, promoting orderly cell arrangement and the orderly secretion of the extracellular matrix, particularly the orderly deposition of collagen. Through a certain period of cultivation, cell proliferation and growth within the matrix material (hydrogel) are promoted, enhancing cell activity in the skin-like structure. Finally, guided by the ordered structure, skin fibroblasts secrete orderly arranged extracellular matrix, especially collagen, constructing a skin-like structure.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] This invention provides a method for constructing ordered skin-like tissue with magnetic field assistance. The method involves preparing ordered hydrogels based on directional cryocasting, co-culturing magnetized cells with the ordered hydrogel under a magnetic field to obtain a hydrogel scaffold, and simultaneously seeding human keratinocytes onto the surface of the hydrogel scaffold. Continuous culturing yields ordered skin-like tissue. The method includes the following steps:

[0010] S1: Directional cryocasting to prepare layered ordered hydrogels, which are used as matrix materials for skin-like tissues;

[0011] S2: Provide skin-like seed cells, and culture and expand them in large quantities using complete culture medium to obtain skin-like cells;

[0012] S3: Skin-like cells cultured in S2 were co-cultured with iron oxide nanoparticles to prepare magnetized skin-like cells.

[0013] S4: The magnetized skin cells prepared in S3 were co-cultured with the layered ordered hydrogel prepared in S1, and magnetic fields were applied on both sides to induce the magnetized skin cells to enter the layered ordered hydrogel, thus obtaining a hydrogel scaffold.

[0014] S5: After washing the hydrogel scaffold obtained in S4, transfer it to a complete culture medium, and simultaneously inoculate keratinocytes on the surface of the hydrogel scaffold, culture, and prepare an ordered structured skin-like tissue.

[0015] In one embodiment of the present invention, in step S1, the layered ordered hydrogel is formed by photocured GelMA through directional cryogenic casting; the preparation method specifically includes the following steps:

[0016] S11: Prepare GelMA solution by dissolving GelMA and LAP photoinitiator in PBS, vortexing in the dark, and then incubating in the dark on a shaker.

[0017] S12: Prepare GelMA cryogenic precursor by adding GelMA solution into a mold and storing it at 4°C in the dark to form GelMA cryogenic precursor;

[0018] S13: Add liquid nitrogen into the foam box and cover it with a thermally conductive copper sheet. Then, invert the mold containing the GelMA cryogenic precursor obtained in step S12 onto the copper sheet for cryogenic casting. Irradiate the cryogenically cast hydrogel under a light source to gel it, and quickly transfer it to a freeze dryer for freeze drying to obtain a layered ordered hydrogel.

[0019] In one embodiment of the present invention, more specifically, in step S1, the layered ordered hydrogel is formed by directional cryogenic casting of GelMA; the preparation method specifically includes the following steps:

[0020] S11: Prepare GelMA solution by dissolving GelMA and LAP photoinitiator in PBS, vortexing in the dark for 5 minutes, and then incubating in a shaker at 37°C for 0.1-24 hours in the dark. The ratio of GelMA, LAP photoinitiator, and PBS should be scaled up or reduced as follows: 10-1400 mg of GelMA and 0.1-50 mg of LAP photoinitiator dissolved in 10 mL of PBS.

[0021] S12: Prepare GelMA cryogenic precursor by adding an appropriate amount of GelMA solution into a mold and storing it at 4°C in the dark for 1-100 minutes to form GelMA cryogenic precursor.

[0022] S13: Freeze casting of GelMA cryogenic precursor. Liquid nitrogen is added to a foam box and a thermally conductive copper sheet is placed on top. Then, the mold containing the GelMA cryogenic precursor obtained in step S12 is inverted on the copper sheet for freeze casting. The freeze-cast hydrogel is irradiated under a 405 nm light source for 1-600 s to gel it, and then quickly transferred to a freeze dryer for freeze drying.

[0023] In one embodiment of the present invention, in step S2, the skin-like seed cells are selected from one or more of induced human pluripotent stem cells (iPSCs), human epidermal stem cells, animal epidermal stem cells, human skin fibroblasts, animal skin fibroblasts, human keratinocytes, or animal keratinocytes. Preferably, in step S2, the skin-like seed cells are selected from human skin fibroblasts.

[0024] In one embodiment of the present invention, in step S3, when the skin-like cells are co-cultured with iron oxide nanoparticles, the concentrations of the iron oxide nanoparticles and the skin-like cells are 1~1000 μg of iron oxide and 1×10⁻⁶ μg of iron oxide per mL of solution, respectively. 4 ~1×10 7 Each cell.

[0025] In one embodiment of the present invention, in step S3, the co-cultivation time is 0.01 to 72 hours, preferably 12 hours.

[0026] In one embodiment of the present invention, in step S4, the relationship between the amount of magnetized skin cells and the layered ordered hydrogel is as follows: the amount of magnetized skin cells is 5 × 10⁻⁶. 5 Individuals were co-cultured with ordered hydrogels measuring 10 mm × 10 mm × 2 mm under a magnetic field of 0.4–0.6 T.

[0027] In one embodiment of the present invention, in step S4, the co-cultivation time is 1 to 30 days, preferably 2 days.

[0028] In one embodiment of the present invention, in step S5, the culture conditions are: culture for 0.1-60 days, and fresh culture medium is used every 2-3 days during the culture period.

[0029] In one embodiment of the present invention, in step S5, the method for preparing the complete culture medium is to add 50 mL of FBS and 5 mL of PS to 500 mL of basal culture medium; the basal culture medium used includes DMEM.

[0030] The present invention further provides an ordered structured skin-like tissue, which is prepared by the above method.

[0031] The solution provided in this application shifts the focus from "cell construction" in existing technologies to "structure-function integrated biomimicry." This application provides a method for introducing multi-layered, orderly arranged tissue structures into skin-like structures to more accurately simulate the physiological state of natural skin, thereby enhancing its application value in complex wound repair. In the solution provided in this application, the ordered structure and bioactivity facilitate the adaptive integration of the skin-like structure with the defect site and induce orderly ingrowth of cells in vivo, achieving scarless wound healing.

[0032] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0033] 1. In this invention, magnetized fibroblasts are generated using iron oxide nanoparticles, and the fibroblasts are guided into the ordered channels of the scaffold material under the induction of an external magnetic field.

[0034] 2. This invention utilizes a hydrogel scaffold material with a layered, ordered structure and interconnected pores for skin-like culture, effectively regulating the migration and arrangement of fibroblasts. The orderly arrangement of fibroblasts in the matrix material (hydrogel scaffold material) provides a physical basis for the arrangement of the extracellular matrix, especially collagen. Attached Figure Description

[0035] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0036] Figure 1 This is a flowchart of the method for constructing ordered skin-like tissue with magnetic field assistance according to the present invention.

[0037] Figure 2 These are photographs of the gelatinized gelatin and ordered methacrylated gelatin provided in Example 1 of the present invention.

[0038] Figure 3 These are scanning electron microscope (SEM) images of methacrylated gelatin and ordered methacrylated gelatin hydrogel before and after cryocasting, as provided in Example 1 of this invention.

[0039] Figure 4 This is a CCK-8 result diagram showing the effect of the hydrogel scaffold on fibroblast proliferation in two-dimensional culture in Embodiment 1 of the invention.

[0040] Figure 5 Transmission electron microscopy image of iron oxide nanoparticles entering fibroblasts.

[0041] Figure 6 The images show the distribution of iron oxide nanoparticles under a magnetic field and the cell arrangement after magnetization of iron oxide.

[0042] Figure 7 A three-dimensional skeletal staining diagram of cells in a skin-like structure. Detailed Implementation

[0043] refer to Figure 1 This invention provides a method for constructing ordered skin-like tissue with magnetic field assistance. The method involves preparing ordered hydrogels based on directional cryocasting, co-culturing magnetized cells with the ordered hydrogel under a magnetic field to obtain a hydrogel scaffold, and simultaneously seeding human keratinocytes onto the surface of the hydrogel scaffold. Continuous culturing yields ordered skin-like tissue. The method includes the following steps:

[0044] S1: Directional cryocasting to prepare layered ordered hydrogels, which are used as matrix materials for skin-like tissues;

[0045] S2: Provide skin-like seed cells, and culture and expand them in large quantities using complete culture medium to obtain skin-like cells;

[0046] S3: The skin-like cells cultured in S2 were co-cultured with iron oxide nanoparticles to prepare magnetized skin-like cells;

[0047] S4: The magnetized skin cells prepared in S3 were co-cultured with the layered ordered hydrogel prepared in S1, and magnetic fields were applied on both sides to induce the magnetized skin cells to enter the layered ordered hydrogel, thus obtaining a hydrogel scaffold.

[0048] S5: After washing the hydrogel scaffold obtained in S4, transfer it to a complete culture medium and simultaneously inoculate keratinocytes on the surface of the hydrogel scaffold to prepare an ordered skin-like tissue.

[0049] In one embodiment of the present invention, in step S1, the layered ordered hydrogel is formed by photocured GelMA through directional cryogenic casting; the preparation method specifically includes the following steps:

[0050] S11: Prepare GelMA solution by dissolving GelMA and LAP photoinitiator in PBS, vortexing in the dark, and then incubating in the dark on a shaker.

[0051] S12: Prepare GelMA cryogenic precursor by adding GelMA solution into a mold and storing it at 4°C in the dark to form GelMA cryogenic precursor;

[0052] S13: Add liquid nitrogen into the foam box and cover it with a thermally conductive copper sheet. Then, invert the mold containing the GelMA cryogenic precursor obtained in step S12 onto the copper sheet for cryogenic casting. Irradiate the cryogenically cast hydrogel under a light source to gel it, and quickly transfer it to a freeze dryer for freeze drying to obtain a layered ordered hydrogel.

[0053] In one embodiment of the present invention, more specifically, in step S1, the layered ordered hydrogel is formed by directional cryogenic casting of GelMA; the preparation method specifically includes the following steps:

[0054] S11: Prepare GelMA solution by dissolving GelMA and LAP photoinitiator in PBS, vortexing in the dark for 5 minutes, and then incubating in a shaker at 37°C for 0.1-24 hours in the dark. The ratio of GelMA, LAP photoinitiator, and PBS should be scaled up or reduced as follows: 10-1400 mg of GelMA and 0.1-50 mg of LAP photoinitiator dissolved in 10 mL of PBS.

[0055] S12: Prepare GelMA cryogenic precursor by adding an appropriate amount of GelMA solution into a mold and storing it at 4°C in the dark for 1-100 minutes to form GelMA cryogenic precursor.

[0056] S13: Freeze casting of GelMA cryogenic precursor. Liquid nitrogen is added to a foam box and a thermally conductive copper sheet is placed on top. Then, the mold containing the GelMA cryogenic precursor obtained in step S12 is inverted on the copper sheet for freeze casting. The freeze-cast hydrogel is irradiated under a 405 nm light source for 1-600 s to gel it, and then quickly transferred to a freeze dryer for freeze drying.

[0057] In one embodiment of the present invention, in step S2, the skin-like seed cells are selected from one or more of induced human pluripotent stem cells (iPSCs), human epidermal stem cells, animal epidermal stem cells, human skin fibroblasts, animal skin fibroblasts, human keratinocytes, or animal keratinocytes. Preferably, in step S2, the skin-like seed cells are selected from human skin fibroblasts.

[0058] In one embodiment of the present invention, in step S3, when the skin-like cells are co-cultured with iron oxide nanoparticles, the concentrations of the iron oxide nanoparticles and the skin-like cells are 1~1000 μg of iron oxide and 1×10⁻⁶ μg of iron oxide per mL of solution, respectively. 4 ~1×10 7 Each cell.

[0059] In one embodiment of the present invention, in step S3, the co-cultivation time is 0.01 to 72 hours, preferably 12 hours.

[0060] In one embodiment of the present invention, in step S4, the amount of magnetized skin cells and the layered ordered hydrogel are related as follows: the amount of magnetized skin cells is 5 × 10⁻⁶. 5 Individuals were co-cultured with ordered hydrogels measuring 10 mm × 10 mm × 2 mm under a magnetic field of 0.4–0.6 T.

[0061] In one embodiment of the present invention, in step S4, the co-cultivation time is 1 to 30 days, preferably 2 days.

[0062] In one embodiment of the present invention, in step S5, the culture conditions are: culture for 0.1-60 days, preferably 6-7 days, and the culture medium is replaced with fresh culture medium every 2-3 days during the culture period.

[0063] In one embodiment of the present invention, in step S5, the method for preparing the complete culture medium is to add 50 mL of FBS and 5 mL of PS to 500 mL of basal culture medium; the basal culture medium used includes DMEM.

[0064] The present invention will be further described below with reference to specific embodiments.

[0065] Example 1

[0066] Reference Figures 2 to 7 This embodiment provides a method for constructing ordered skin-like tissue with magnetic field assistance. Based on directional cryo-casting to prepare ordered hydrogels, a large number of expanded magnetized human skin fibroblasts are co-cultured with the hydrogel under the influence of a magnetic field. Simultaneously, human keratinocytes are seeded on the surface, and the mixture is continuously cultured for 14 days to form skin-like tissue. The method includes the following steps:

[0067] S1: Preparation of skin-like matrix material, layered ordered hydrogel;

[0068] S2: Provides human skin fibroblasts, which are cultured and expanded in large quantities using complete culture medium;

[0069] S3: Preparation of magnetized human skin fibroblasts. The human skin fibroblasts obtained in S2 were co-cultured with iron oxide nanoparticles for 24 hours to prepare magnetized human skin fibroblasts. The concentrations of iron oxide nanoparticles and human skin fibroblasts were 100 μg iron oxide and 1×10⁻⁶ iron oxide per mL of solution, respectively. 6 One cell;

[0070] S4: Magnetic field guides cells into the material prepared in S1. Magnetized human skin fibroblasts prepared in S3 and the layered ordered hydrogel prepared in S1 are co-cultured in complete culture medium, and magnetic fields are applied on both sides for induction. The ratio of magnetized human skin fibroblasts to layered ordered hydrogel is 5 × 10⁻⁶. 5 A layered ordered hydrogel of 10 mm × 10 mm × 2 mm (approximately 0.2 g) was co-cultured with 2 ml of complete culture medium at 4 °C under a magnetic field of 0.5 T for 2 days in an incubator to culture the skin-like material.

[0071] S5: After washing the sample obtained in S4, transfer it to complete culture medium. The complete culture medium is prepared by adding 50 mL of FBS and 5 mL of PS to 500 mL of basal culture medium. The basal culture medium used is DMEM. At the same time, keratinocytes are seeded on the scaffold surface and cultured for 14 days. Fresh culture medium is used every 3 days during the culture period to prepare a skin-like structure with an ordered structure.

[0072] In this embodiment, the layered ordered hydrogel is formed by directional cryogenic casting of GelMA; the specific preparation method of the layered ordered hydrogel is as follows:

[0073] S11: Prepare GelMA solution by dissolving 700 mg of GelMA and 25 mg of LAP photoinitiator in 10 mL of PBS, vortexing in the dark for 5 minutes, and then incubating in the dark at 37°C for 12 hours.

[0074] S12: Prepare GelMA cryogenic precursor by adding an appropriate amount of GelMA solution into a mold and storing it at 4°C in the dark for 30 minutes to form GelMA cryogenic precursor.

[0075] S13: Cryogenic casting of GelMA cryogenic precursor. Liquid nitrogen was added to a foam box and a thermally conductive copper sheet was placed on top. The frozen mold was then inverted on the copper sheet for cryogenic casting for 3 hours. The cryogenically cast sample was irradiated under a 405 nm light source for 60 s to gel it and then quickly transferred to a freeze dryer for freeze drying for 3 days.

[0076] This embodiment provides a specific method for preparing layered ordered hydrogels. For comparison, ordinary methacrylated gelatin is also provided. The preparation method of ordinary methacrylated gelatin is as follows: S11: Prepare GelMA solution by dissolving 700 mg of GelMA and 25 mg of LAP photoinitiator in 10 mL of PBS, vortexing in the dark for 5 minutes, and then incubating in a shaker at 37°C for 12 hours in the dark; S12: Prepare GelMA cryogenic precursor by adding an appropriate amount of GelMA solution to a mold; irradiate under a 405 nm light source for 60 s to gel, and then rapidly transfer to liquid nitrogen for 6 hours of quick-freezing; S13: Rapidly transfer the sample in liquid nitrogen to a freeze dryer and dry for 3 days. Physical images of the layered ordered hydrogel (i.e., ordered methacrylated gelatin) and ordinary methacrylated gelatin are shown below. Figure 2 As shown, the layered ordered hydrogel produced by this method exhibits white bubble-like dots but remains in a colloidal state, appearing transparent.

[0077] Scanning electron micrographs of layered ordered hydrogels (i.e., ordered methacrylated gelatin) and ordinary methacrylated gelatin are shown below. Figure 3 As shown, ordinary methacrylated gelatin has the typical porous structure of hydrogels, while layered ordered hydrogels (i.e., ordered methacrylated gelatin) exhibit a layered ordered structure and continuous pores, which is conducive to cell proliferation and migration and the orderly precipitation of extracellular matrix.

[0078] In this embodiment, transmission electron microscopy (TEM) images of fibroblasts taking up iron oxide nanoparticles were obtained after S3 treatment. Figure 5 As shown, fibroblasts successfully took up iron oxide nanoparticles to form the basis for magnetic field-guided cell arrangement.

[0079] Figure 6 The arrangement of iron oxide nanoparticles and magnetized cells under the influence of a magnetic field is shown. Both the iron oxide nanoparticles and magnetized cells are distributed according to the magnetic field.

[0080] Figure 7 The top image shows a three-dimensional cytoskeleton staining diagram of the skin-like cells in this culture method. It can be seen that the cells are arranged in an orderly manner along the hydrogel pores.

[0081] Comparative Example 1:

[0082] Comparative Example 1 provides a method for constructing ordered skin-like tissue with magnetic field assistance. Based on a hydrogel prepared without directional cryo-casting (i.e., ordinary methacrylated gelatin), a large number of expanded magnetized human skin fibroblasts are co-cultured with the hydrogel (i.e., ordinary methacrylated gelatin) under the influence of a magnetic field. Simultaneously, human keratinocytes are seeded onto the surface, and the mixture is cultured continuously for 14 days to form skin-like tissue. The difference between this example and Example 1 is that the matrix material used to prepare the skin-like tissue is a hydrogel prepared without directional cryo-casting (i.e., ordinary methacrylated gelatin).

[0083] The specific steps are as follows:

[0084] S1: Preparation of skin-like matrix material hydrogel;

[0085] S2: Provides skin-like seed cells, which are then cultured and expanded in large quantities using complete culture medium;

[0086] S3: Prepare magnetized skin fibroblasts by co-culturing the skin fibroblasts obtained in S2 with iron oxide nanoparticles for 12 hours.

[0087] S4: A magnetic field guides cells into the material prepared in S1. Cells prepared in S3 are co-cultured with the sample prepared in S1, and magnetic fields are applied to both sides for induction. The skin-like material is then cultured in an incubator for 2 days.

[0088] S5: After washing the sample obtained in S4, transfer it to a complete culture medium. At the same time, inoculate keratinocytes on the scaffold surface and culture for 14 days. During the culture period, replace the culture medium with fresh medium every 2-3 days to prepare a skin-like structure.

[0089] The hydrogel described in S1 is prepared directly from GelMA, and the specific method is as follows:

[0090] S11: Prepare GelMA solution by dissolving 700 mg of GelMA and 25 mg of LAP photoinitiator in 10 mL of PBS, vortexing in the dark for 5 minutes, and then incubating in the dark at 37°C for 12 hours.

[0091] S12: Prepare a GelMA cryogenic precursor by adding an appropriate amount of GelMA solution into a mold; irradiate it under a 405 nm light source for 60 s to gel it, and then quickly transfer it to liquid nitrogen for 6 hours for rapid freezing.

[0092] S13: Quickly transfer the sample in liquid nitrogen to a freeze dryer and dry for 3 days.

[0093] Except for the hydrogel used, which is different from that in Example 1, all other experimental conditions in Comparative Example 1 are the same as in Example 1.

[0094] Figure 4 CCK-8 results show the effects of different hydrogel scaffolds on fibroblast proliferation in two-dimensional culture. Figure 4 In the text, "ns" indicates no statistical difference, "*" indicates a statistical difference (p<0.05), and "***" indicates a statistically significant difference (p<0.001).

[0095] The CCK test method for ordered methacrylated gelatin is as follows: 0.2 g of ordered hydrogel (the layered ordered hydrogel in Example 1) was soaked in 2 ml of complete culture medium at 4°C for 24 hours to prepare an extract. 5000 skin fibroblasts were cultured for 24 hours and then incubated with 200 μL of the extract in a 96-well plate for 24 hours. The biocompatibility was detected using CCK reagent.

[0096] The CCK assay for methacrylated gelatin was performed as follows: 0.2 g of methacrylated gelatin (ordinary methacrylated gelatin from Comparative Example 1) was soaked in 2 ml of complete culture medium at 4°C for 24 hours to prepare an extract. 5000 skin fibroblasts were cultured for 24 hours, and then co-incubated with 200 μL of the extract in a 96-well plate for 24 hours. Biocompatibility was assessed using the CCK reagent. Cell viability was shown in the figure. Figure 4 ;

[0097] The CCK assay for iron(III) oxide was performed as follows: 5000 skin fibroblasts were cultured for 24 hours, followed by co-incubation with 200 μL of complete culture medium containing 100 ng / ml iron(III) oxide for 24 hours. Biocompatibility was then assessed using the CCK reagent. Cell viability was recorded as follows: Figure 4 ;

[0098] The control CCK assay involved culturing 5000 skin fibroblasts for 24 hours, followed by co-incubation with 200 μL of complete culture medium for another 24 hours. Biocompatibility was then assessed using CCK reagent, and cell viability was recorded. Figure 4 ;

[0099] These experiments were identical except for the conditions under which the cells were co-incubated.

[0100] pass Figure 4 The first and third bars show that there was no significant difference in fibroblast proliferation in the GelMA group, indicating that the hydrogel has good biocompatibility. The fourth bar shows that the iron oxide nanoparticles do not significantly affect fibroblast activity.

[0101] Example 2

[0102] A large number of expanded magnetized human skin fibroblasts were co-cultured with hydrogel under a magnetic field, and human keratinocytes were simultaneously seeded on the surface. The cells were cultured continuously for 14 days to form a skin-like structure. The difference between this embodiment and Example 1 is that the co-culture time of hydrogel and cells in S5 is 8 days. The specific method of this embodiment includes the following steps:

[0103] S1: Preparation of skin-like matrix material, layered ordered hydrogel;

[0104] S2: Provides skin-like seed cells, which are then cultured and expanded in large quantities using complete culture medium;

[0105] S3: Prepare magnetized skin fibroblasts by co-culturing the skin fibroblasts obtained in S2 with iron oxide nanoparticles for 12 hours.

[0106] S4: A magnetic field guides cells into the material prepared in S1. Cells prepared in S3 are co-cultured with the sample prepared in S1, and magnetic fields are applied to both sides for induction. The skin-like material is then cultured in an incubator for 2 days.

[0107] S5: After washing the sample obtained in S4, transfer it to a complete culture medium. At the same time, inoculate keratinocytes on the scaffold surface and culture for 8 days. During the culture period, replace the culture medium with fresh medium every 3 days to prepare a skin-like structure with an ordered structure.

[0108] The layered ordered hydrogel described in S1 is formed by directional cryogenic casting of GelMA; the specific preparation method of the layered ordered hydrogel is as follows:

[0109] S11: Prepare GelMA solution by dissolving 700 mg of GelMA and 25 mg of LAP photoinitiator in 10 mL of PBS, vortexing in the dark for 5 minutes, and then incubating in the dark at 37°C for 12 hours.

[0110] S12: Prepare GelMA cryogenic precursor by adding an appropriate amount of GelMA solution into a mold and storing it at 4°C in the dark for 30 minutes to form GelMA cryogenic precursor.

[0111] S13: Cryogenic casting of GelMA cryogenic precursor. Liquid nitrogen was added to a foam box and a thermally conductive copper sheet was placed on top. The frozen mold was then inverted on the copper sheet for cryogenic casting for 3 hours. The cryogenically cast sample was irradiated under a 405 nm light source for 60 s to gel it and then quickly transferred to a freeze dryer for freeze drying for 3 days.

[0112] Example 3

[0113] A large number of expanded human skin fibroblasts were co-cultured with hydrogel under the action of a magnetic field, and human keratinocytes were seeded on the surface. The cells were cultured continuously for 14 days to form skin-like cells. The difference between this example and Example 1 is that the cells used in the construction of the skin-like cells were not magnetized with iron oxide.

[0114] S1: Preparation of skin-like matrix material, layered ordered hydrogel;

[0115] S2: Provides skin-like seed cells, which are then cultured and expanded in large quantities using complete culture medium;

[0116] S3: Cells enter the material prepared in S1 under the influence of a magnetic field. Cells prepared in S2 are co-cultured with the sample prepared in S1, and magnetic fields are applied to both sides for induction. The mixture is then incubated for 12 hours.

[0117] S5: After washing the sample obtained in S4, transfer it to a complete culture medium. At the same time, inoculate keratinocytes on the scaffold surface and culture for 14 days. During the culture period, replace the culture medium with fresh medium every 3 days to prepare a skin-like structure with an ordered structure.

[0118] The layered ordered hydrogel described in S1 is formed by directional cryogenic casting of GelMA; the specific preparation method of the layered ordered hydrogel is as follows:

[0119] S11: Prepare GelMA solution by dissolving 700 mg of GelMA and 25 mg of LAP photoinitiator in 10 mL of PBS, vortexing in the dark for 5 minutes, and then incubating in the dark at 37°C for 12 hours.

[0120] S12: Prepare GelMA cryogenic precursor by adding an appropriate amount of GelMA solution into a mold and storing it at 4°C in the dark for 30 minutes to form GelMA cryogenic precursor.

[0121] S13: Cryogenic casting of GelMA cryogenic precursor. Liquid nitrogen was added to a foam box and a thermally conductive copper sheet was placed on top. The frozen mold was then inverted on the copper sheet for cryogenic casting for 3 hours. The cryogenically cast sample was irradiated under a 405 nm light source for 60 s to gel it and then quickly transferred to a freeze dryer for freeze drying for 3 days.

[0122] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for constructing ordered skin-like tissue with magnetic field assistance, characterized in that, Includes the following steps: S1: Directional cryocasting to prepare layered ordered hydrogels, which are used as matrix materials for skin-like tissues; S2: Provide skin-like seed cells, and culture and expand them in large quantities using complete culture medium to obtain skin-like cells; S3: Skin-like cells cultured in S2 were co-cultured with iron oxide nanoparticles to prepare magnetized skin-like cells. S4: The magnetized skin cells prepared in S3 were co-cultured with the layered ordered hydrogel prepared in S1, and magnetic fields were applied on both sides to induce the magnetized skin cells to enter the layered ordered hydrogel, thus obtaining a hydrogel scaffold. S5: After washing the hydrogel scaffold obtained in S4, transfer it to a complete culture medium, and simultaneously inoculate keratinocytes on the surface of the hydrogel scaffold, culture, and prepare an ordered structured skin-like tissue. In step S1, the layered ordered hydrogel is formed by photocured GelMA through directional cryocasting; the preparation method specifically includes the following steps: S11: Prepare GelMA solution by dissolving GelMA and LAP photoinitiator in PBS, vortexing in the dark, and then incubating in the dark on a shaker. S12: Prepare GelMA cryogenic precursor by adding GelMA solution into a mold and storing it at 4°C in the dark to form GelMA cryogenic precursor; S13: Add liquid nitrogen into the foam box and cover it with a thermally conductive copper sheet. Then, invert the mold containing the GelMA cryogenic precursor obtained in step S12 onto the copper sheet for cryogenic casting. Irradiate the cryogenically cast hydrogel under a light source to gel it, and quickly transfer it to a freeze dryer for freeze drying to obtain a layered ordered hydrogel.

2. The method for constructing ordered skin-like tissue with magnetic field assistance according to claim 1, characterized in that, In step S1, the layered ordered hydrogel is formed by directional cryogenic casting of GelMA; the preparation method specifically includes the following steps: S11: Prepare GelMA solution by dissolving GelMA and LAP photoinitiator in PBS, vortexing in the dark for 5 minutes, and then incubating in a shaker at 37°C for 0.1-24 hours in the dark. The ratio of GelMA, LAP photoinitiator, and PBS should be scaled up or reduced as follows: 10-1400 mg of GelMA and 0.1-50 mg of LAP photoinitiator dissolved in 10 mL of PBS. S12: Prepare GelMA cryogenic precursor by adding an appropriate amount of GelMA solution into a mold and storing it at 4°C in the dark for 1-100 minutes to form GelMA cryogenic precursor. S13: Freeze casting of GelMA cryogenic precursor. Liquid nitrogen is added to a foam box and a thermally conductive copper sheet is placed on top. Then, the mold containing the GelMA cryogenic precursor obtained in step S12 is inverted on the copper sheet for freeze casting. The freeze-cast hydrogel is irradiated under a 405 nm light source for 1-600 s to gel it, and then quickly transferred to a freeze dryer for freeze drying.

3. The method for constructing ordered skin-like tissue with magnetic field assistance according to claim 1, characterized in that, In step S2, the skin-like seed cells are selected from one or more of induced human pluripotent stem cells, human epidermal stem cells, animal epidermal stem cells, human skin fibroblasts, animal skin fibroblasts, human keratinocytes, or animal keratinocytes.

4. The method for constructing ordered skin-like tissue with magnetic field assistance according to claim 1, characterized in that, In step S3, when skin-like cells are co-cultured with iron oxide nanoparticles, the concentrations of iron oxide nanoparticles and skin-like cells are 1–1000 μg of iron oxide and 1 × 10⁻⁶ μg of iron oxide per mL of solution, respectively. 4 ~1×10 7 Each cell.

5. The method for constructing ordered skin-like tissue with magnetic field assistance according to claim 1, characterized in that, In step S4, the ratio of the amount of magnetized skin cells to the amount of layered ordered hydrogel is as follows: the amount of magnetized skin cells is 5 × 10⁻⁶. 5 Each was co-cultured with an ordered hydrogel measuring 10 mm × 10 mm × 2 mm.

6. The method for constructing ordered skin-like tissue with magnetic field assistance according to claim 1, characterized in that, In step S4, the magnetic field size is 0.4~0.6T.

7. The method for constructing ordered skin-like tissue with magnetic field assistance according to claim 1, characterized in that, In step S5, the culture conditions are: culture for 0.1-60 days, and fresh culture medium is used every 2-3 days during the culture period.

8. The method for constructing ordered skin-like tissue with magnetic field assistance according to claim 1, characterized in that, In step S5, the complete culture medium is prepared by adding 50 mL of FBS and 5 mL of PS to 500 mL of basal culture medium; the basal culture medium used includes DMEM.

9. An ordered structured skin-like tissue, characterized in that, Prepared by the method described in any one of claims 1-8.

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