Preparation method of prefabricated biological 3D printing artificial skin for skin and hair follicle regeneration

Abstract

The invention provides a preparation method of prefabricated biological 3D printing artificial skin for skin and hair follicle regeneration. The prefabricated biological 3D printing artificial skin mainly comprises GelMA, HAMA and skin stem cells. The preparation method of the biological 3D printing artificial skin comprises the steps that a hydrogel solution containing 50 mg of GelMA and 5 mg of HAMA per milliliter is prepared firstly, then 107-108 skin stem cells are added, and the proportion of dermal stem cells to epidermal stem cells is 1: 2. And finally, mixing the cells with the hydrogel, transferring into an injector, refrigerating for 3 minutes, transferring into a biological 3D printer, and preparing artificial skins of different models according to preset printing parameters. The optimized hydrogel material disclosed by the invention has excellent printability, relatively low solubility and swelling rate and stable mechanical properties. The hydrogel material can be matched with the skin stem cells to promote the dryness of the skin stem cells to be improved, and skin repair and hair follicle regeneration are better facilitated. The biological 3D printing artificial skin prepared by the invention can be used for promoting skin and hair follicles in wounds immediately or after in-vitro culture.

Description

Technical Field

[0001] The present invention belongs to the fields of regenerative medicine and tissue engineering, and particularly relates to a method for preparing a prefabricated bio-3D printed artificial skin for skin and hair follicle regeneration. Background Art

[0002] The skin, together with sweat glands, sebaceous glands, hair follicles and other accessory organs, constitutes a continuous external barrier system and is one of the most important organs of the human body. The skin has biological functions such as resisting the invasion of foreign bodies, regulating body temperature and preventing water loss. Wounds are caused by external trauma or pathogenic factors that damage the integrity of the skin, including surgical operations, thermal injuries, electric burns and stress-related injuries encountered in daily life. Wounds can cause pain, anxiety, infection and even death, seriously damaging the quality of life of patients and bringing a considerable burden to the healthcare system at the same time.

[0003] Currently, the traditional treatment methods for large-area skin wounds mainly rely on autologous skin transplantation. However, this technology has drawbacks such as secondary pain, limited donor sites and high infection risks. To solve the above problems, using tissue engineering to prepare skin substitutes has become a new idea. Hydrogels are widely used in the field of skin repair due to their excellent water absorption capacity, moisturizing performance, biocompatibility and three-dimensional porous structure. Selecting appropriate hydrogel materials and seed cells is crucial for the regeneration of the skin and its accessory organs. In the prior art, combining hydrogel materials with drugs or cells can promote wound healing, but it is still inadequate in the regeneration of accessory organs such as hair follicles, sebaceous glands and sweat glands. A number of known technologies use artificial skin prepared by combining dermal papilla cells (DPCs) of hair follicles with hydrogel materials and have the ability to induce hair regeneration. However, DPCs have limitations such as scarce quantity, difficult acquisition and difficulty in maintaining the hair follicle regeneration potential during in vitro culture. In addition, the development of engineered and intelligent artificial skin is also crucial for large-scale clinical applications. In this regard, bio-3D printing technology provides an efficient and automated way to print artificial skin in various forms. Bio-3D printed artificial skin can establish appropriate intercellular connections by precisely controlling the number of printed skin layers and cell density, thereby promoting the paracrine effect and interaction between cells within the tissue. In addition, due to the immaturity of the in vitro culture technology of artificial skin, large-area trauma patients in clinical settings cannot receive immediate treatment. Therefore, developing a prefabricated artificial skin that can adapt to bio-3D printing and achieve the regeneration of the skin and its accessory organs has significant practical significance. Summary of the Invention

[0004] The object of the present invention is to provide a method for preparing a prefabricated bio-3D printed artificial skin for skin and hair follicle regeneration to solve the existing problems in view of the technical problems in the background art.

[0005] The technical solution adopted by the present invention is as follows:

[0006] A method for preparing a prefabricated bio-3D printed artificial skin for skin and hair follicle regeneration, comprising the following steps:

[0007] S1: Prepare the hydrogel material for the bio-3D printed artificial skin. Each milliliter of the hydrogel material contains 50 mg of methacrylated gelatin (GelMA), 5 mg of methacrylated hyaluronic acid (HAMA), and 5 mg of lithium phenyl-2,4,6-trimethylbenzoylphosphate (LAP) photoinitiator;

[0008] S2: Debug the bio-3D printing model of the hydrogel material in step S1 to determine the printer parameters;

[0009] S3: Add skin stem cells to the hydrogel material prepared in step S1, with 10 7 ~10 8 skin stem cells per milliliter; S4: Transfer the multi-component hydrogel material prepared in step S3 into a syringe, pre-cool it in the refrigerator for 3 minutes, and then transfer it into a bio-3D printer. Use the printing parameters determined in step S2 to prepare the bio-3D printed artificial skin;

[0010] S5: Perform UV cross-linking molding on the bio-3D printed artificial skin prepared in step S4 and culture it in vitro.

[0011] Further preferably, in the above step S2, the printer parameters are: nozzle aperture 0.45 mm, nozzle extrusion distance 0.1 mm, layer-by-layer printing height 0.15 mm, and nozzle moving speed 8 mm / s.

[0012] Further preferably, in the above step S3, the skin stem cells include epidermal stem cells and dermal stem cells; the ratio of the number content of the epidermal stem cells to the dermal stem cells is 1:2.

[0013] Further preferably, in the above step S5, the specific steps are as follows: First, perform ultraviolet light irradiation treatment on the bio-3D printed artificial skin, with the distance from the ultraviolet lamp being 10-20 cm and the irradiation time being 150-300 s.

[0014] Further preferably, after the artificial skin is cured in the above step S5, it is transferred into a CnT-Prime 3D Barrier special medium and cultured in vitro in a 37°C constant temperature incubator.

[0015] Further preferably, the above epidermal stem cells are epidermal stem cells obtained by shearing and separating skin tissues, digesting with proteolytic dispersing enzyme and collagenase, screening and collecting cells, and finally performing adherent culture using Keratinocyte-SFM epidermal keratinocyte medium.

[0016] More preferably, the above-mentioned dermal stem cells are obtained by shearing and separating skin tissue, digesting with protein dispersing enzyme and collagenase, screening and collecting cells, and finally culturing in DMEM / F12 medium containing B27, EGF and bFGF. More preferably, the above-mentioned method for preparing a prefabricated bio-3D printed artificial skin for skin and hair follicle regeneration can be used to promote the regeneration of wound skin and hair follicles.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] (1) The present invention selects GelMA and HAMA hydrogels as bioactive materials for preparing artificial skin, which have low swelling degree and solubility, excellent printing performance and stable mechanical properties, and are suitable for constructing bio-3D printed artificial skin. In addition, the selected hydrogel can be cross-linked and cured by UV, with the advantages of fast gel formation and simple operation.

[0019] (2) The present invention selects epidermal stem cells and dermal stem cells as seed cells. These two types of cells have a wide source and relatively simple separation methods. In addition, dermal stem cells can promote hair follicle regeneration, while epidermal stem cells promote the regeneration of other skin appendages such as sebaceous glands and sweat glands through interaction with dermal stem cells.

[0020] (3) The hydrogel material selected by the present invention has pore size and stiffness suitable for skin regeneration, and can cooperate with skin stem cells to maintain the proliferation and vitality of skin stem cells, improve the stemness of dermal stem cells and the potential of hair follicle regeneration, and is more conducive to skin repair and hair follicle regeneration.

[0021] (4) The bio-3D printed artificial skin prepared by the present invention can achieve skin and hair follicle regeneration after being cultured in vitro for more than ten hours, which is beneficial for immediate clinical application. Description of the Drawings

[0022] Figure 1 It is a characterization and evaluation diagram of the printability of multi-component hydrogels: (A) Scanning electron microscope image of the hydrogel material; (B) Construction of various bio-3D printing models; (C) Realization of grid bio-3D layer-by-layer printing; (D) Macroscopic image of the bio-3D printed hydrogel material; (E) Quantitative analysis of the printability of the hydrogel material; (F) Solubility detection of the hydrogel material; (G) Swelling rate detection of the hydrogel material; (H) Rheological property detection of the hydrogel material.

[0023] Figure 2Proliferation and viability of SKPs in multi-component hydrogels: (A) Schematic diagram of the 3D bioprinting process of hydrogel materials mixed with dermal stem cells; (B) Live / dead staining images of dermal stem cells cultured in hydrogel materials for 1 day and 3 days; (C) Quantitative analysis of cell viability of dermal stem cells cultured in hydrogel materials for 1 day and 3 days; (D) Results of the proliferation experiment of dermal stem cells cultured in hydrogel materials; (E) AP staining results of dermal stem cells cultured in hydrogel materials for 4 days.

[0024] Figure 3 Cytological analysis of stem cells in artificial skin: (A and B) Results of real-time fluorescence quantitative PCR of genes related to the stemness of dermal stem cells; (B) Results of real-time fluorescence quantitative PCR of genes related to hair follicle induction of dermal stem cells; (C and D) Immunofluorescence staining images of representative proteins of dermal stem cells; (E) Flow cytometry detection of representative proteins of epidermal stem cells.

[0025] Figure 4 Artificial skin showing the ability to regenerate skin and its appendages: (A) Skin and hair follicle regeneration after using artificial skin on mouse wounds; (B) Outer and inner surfaces of the regenerated skin tissue; (C) H&E staining revealing the structural characteristics of the regenerated skin tissue; (D) Immunofluorescence staining showing the stratified epidermal regeneration of the regenerated skin tissue; (E) Immunofluorescence staining showing angiogenesis in the regenerated skin tissue; (F) Immunofluorescence staining showing sebaceous gland regeneration in the regenerated skin tissue.

[0026] Figure 5 Preformed artificial skin with the ability to regenerate the epidermis and its appendages: (A) Outer and inner surfaces of the regenerated tissue of preformed artificial skin; (B) H&E staining clarifying the structural characteristics of the regenerated tissue; (C) Statistical data of regenerated hair; (D) Immunofluorescence staining showing the stratified epidermal regeneration of the regenerated skin tissue; (E) Immunofluorescence staining showing angiogenesis in the regenerated skin tissue; (F) Immunofluorescence staining showing sebaceous gland regeneration in the regenerated skin tissue. Detailed implementation manners

[0027] For the convenience of understanding the present invention, the present invention will be described more comprehensively and meticulously below, but the protection scope of the present invention is not limited to the following specific embodiments.

[0028] Unless otherwise defined, all the professional terms used hereinafter have the same meanings as commonly understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention.

[0029] Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0030] Example 1:

[0031] 1. Isolation and culture of skin stem cells:

[0032] Extraction, culture and collection of epidermal stem cells:

[0033] Epi-SCs were isolated from the back of neonatal C57BL / 6J mice at 0 - 3 days old. First, the neonatal mice were soaked in iodophor for 1 minute, then washed 3 times with 75% ethanol, and then washed with PBS. The back skin of the mice was cut and sliced into pieces of 2 - 3 mm 2 in size. It was treated with 0.3% Dispase II at 37 °C for 60 minutes. After manually separating the epidermis and dermis, the dermal tissue was discarded. The separated epidermal tissue was minced and treated with 0.035% Collagenase I at 37 °C for 60 minutes, passed through a 100-mesh filter, the cell suspension was centrifuged (1300 rpm, 25 °C, 5 minutes), washed 2 times with PBS, and the bottom layer was Epi-SCs. The cells were resuspended and seeded into a 10-cm adherent culture dish at a density of 3×10 6 / mL, and cultured with KSFM medium. The medium was changed the next day after the cells adhered, and then changed every three days thereafter. Passage was carried out when the cells showed large-area confluence.

[0034] Extraction, culture and collection of dermal stem cells:

[0035] SKPs were isolated from the back of neonatal C57BL / 6J mice at 0 - 3 days old. First, the neonatal mice were soaked in iodophor for 1 minute, then washed 3 times with 75% ethanol, and then washed with PBS. The back skin of the mice was cut and sliced into pieces of 2 - 3 mm 2 in size. It was treated with 0.3% Dispase II at 37 °C for 60 minutes. After manually separating the epidermis and dermis, the epidermal tissue was discarded. The dermal tissue was minced and treated with 0.35% Collagenase I at 37 °C for 90 minutes until it became homogenized, passed through an 80-mesh filter, the cell suspension was centrifuged (1300 rpm, 25 °C, 5 minutes), washed 2 times with PBS, and the bottom layer was SKPs. The cells were resuspended and seeded into a 10-cm non-adherent culture dish at a density of 3 - 5×106 / mL. It was cultured with DMEM:F12 (3:1) medium containing 2% B27, 20 ng / mL EGF and 40 ng / mL bFGF. The cytokines were supplemented every 3 days, and passage was carried out on the 7th day.

[0036] 2. Screening of bio-3D printing artificial skin materials:

[0037] First, the present invention characterized different concentrations of hydrogel materials in Table 1 and preliminarily screened the concentrations of GelMA and HAMA hydrogels most suitable for constructing bio-3D printed artificial skin.

[0038] Table 1

[0039]

[0040]

[0041] The characterization results are as follows Figure 1 shown. Figure (A) shows that hydrogel materials with different concentrations all exhibit an interconnected three-dimensional porous network structure, and the pore size gradually decreases with the increase in hydrogel concentration. Figures (F) and (G) are the solubility and swelling tests of the hydrogel materials respectively, and the results show that both the swelling ability and solubility of the hydrogel gradually decrease with the increase in concentration. The rheological test results shown in Figure (H) indicate that within the scanning frequency range of 0.1 - 10 rad / s, hydrogel materials with various concentrations can maintain the ability of a stable solid elastic structure, and the hydrogel stiffness is positively correlated with the concentration. Figure (B) shows the successful construction of different 3D models, and Figure (C) shows the successful realization of printing on a bio-3D printer. The macroscopic images of the bio-3D printing of hydrogel materials with different concentrations in Figure (D) and the quantitative printability results shown in Figure (E) indicate that as the hydrogel concentration increases, the printed models become gradually regular. However, at the same time, multi-component hydrogels with too high a concentration will exhibit excessive gelation and thus problems such as bending, stacking, and even breaking of the printed models will occur.

[0042] Subsequently, the present invention detected the effects of hydrogel materials with various concentrations in Table 1 on dermal stem cells, and the results are as follows Figure 2 shown. Figure (A) shows the process flow of the hybrid printing of multi-component gels and dermal stem cells. Figure (B) is the live / dead cell staining map of dermal stem cells cultured in multi-component hydrogels for 1 day and 4 days, and Figure (C) is the quantitative live / dead cell staining, both showing that dermal stem cells can maintain a viable cell rate in the hydrogel material. Figure (D) is the cell proliferation experiment of dermal stem cells in the hydrogel material, and the results show that dermal stem cells can proliferate significantly in the hydrogel material. Figure (E) is the alkaline phosphatase (AP) staining of dermal stem cells in multi-component hydrogels, showing that the hydrogel material can maintain the hair follicle regeneration ability of dermal stem cells. The results show that Figure 2 the various detection results in

[0043] The present invention comprehensively evaluates the above results and concludes that the multi-component hydrogel containing 50 mg GelMA and 5 mg HAMA (5% GelMA - 0.5% HAMA) per milliliter exhibits good swelling and dissolution properties, excellent bio-3D printing performance, and stable rheological properties. In addition, the multi-component hydrogel at this concentration effectively maintains the proliferation and viability of dermal stem cells, supports the morphological expansion of dermal stem cells, and promotes the high expression of AP levels therein. Therefore, the hydrogel material at this concentration is used as the best biomaterial for constructing bio-3D printed artificial skin.

[0044] 3. Preparation of bio-3D printed artificial skin:

[0045] (1) The epidermal stem cells and dermal stem cells cultured in Example 1 were digested and resuspended in PBS. After centrifugation, the supernatant was discarded, and the cell pellet at the bottom was taken to obtain the skin stem cell component.

[0046] (2) Weigh 10 mg of LAP and dissolve it in 1 mL of PBS to prepare a 10 mg / mL LAP solution. Subsequently, 100 mg of GelMA and 10 mg of HAMA were added to the LAP solution together to prepare a hydrogel solution.

[0047] (3) The skin stem cell component obtained in step (1) was evenly mixed with the hydrogel solution obtained in step (2) in a 1:1 ratio, ensuring that each milliliter of the solution contains 50 mg of GelMA, 5 mg of HAMA, 10 6 -10 8 epidermal stem cells and 10 6 -10 8 dermal stem cells, and the ratio of epidermal stem cells to dermal stem cells is 1:2.

[0048] (4) The multi-component hydrogel prepared in step (3) was placed in a syringe and refrigerated for 3 minutes, and then transferred to a bio-3D printer for printing according to the preset model and parameters.

[0049] The present invention detects the protein and gene expression of skin stem cells in the prepared artificial skin, and the results are as Figure 3As shown in the figure, SKP in the figure represents dermal stem cells cultured normally, and SKP-P represents dermal stem cells cultured in artificial skin. Figure (A) shows that the expression of stemness genes Oct4, Sox2, Nanog, and c-Myc of dermal stem cells in artificial skin is significantly increased, indicating that the stemness of dermal stem cells is well maintained in artificial skin. Figure (B) shows that among the genes related to hair induction ability, the expression levels of α-SMA, BMP4, Fibronectin, and Akp2 are significantly increased, and there is no significant change in other genes. Figures (C, D) show that dermal stem cells still highly express their characteristic proteins Nestin, BMP6, and Fibronectin after being cultured in artificial skin. The results of Figure (E) show that there is no significant difference in the marker proteins CD49f and CD29 of epidermal stem cells cultured in artificial skin and the traditional culture group.

[0050] 4. Establishment of in vivo promotion of skin and hair follicle regeneration by bio-3D printed artificial skin:

[0051] In this invention, skin and hair follicle regeneration experiments were carried out in mice. First, a full-thickness wound with a diameter of 5 mm was created on the back of nude mice, and then artificial skin cultured in vitro for different time periods was transplanted into the wound. Then, it was covered with a 3M film and bandaged. Then, the wound skin was fixed and stained at different times to detect the wound healing and hair follicle regeneration conditions, and the detection results are as Figure 4 and Figure 5 shown.

[0052] Figure 4 Figure (A) shows that the bio-3D printed artificial skin completely covers the wound. After 4 weeks, the wound completely heals, accompanied by obvious hair growth. The inner and outer surface diagrams of the regenerated tissue in Figure (B) show that the artificial skin achieves full-thickness healing of the wound and does not cause hyperplasia formation such as teratoma. H&E staining in Figure (C) confirms the occurrence of epidermal, dermal, and hair follicle regeneration in the wound. Immunofluorescence staining in Figure (D) shows that the regenerated skin tissue has a layered epidermal structure, similar to natural skin. Figures (E) and (F) separately show the presence of blood vessels and sebaceous glands in the regenerated skin tissue.

[0053] Figure 5 Figure (A) shows that the bio-3D printed artificial skin cultured in vitro for different times completely covers the wound. After 4 weeks, the wound completely heals, accompanied by obvious hair growth. H&E staining in Figure (B) confirms that the artificial skin cultured in vitro for different times can achieve epidermal, dermal, and hair follicle regeneration in the wound. The number of hairs in the regenerated tissue of the artificial skin cultured in vitro for different time periods was counted in Figure (C), and it was found that the hair regeneration ability gradually decreased with the extension of the culture time. Figure (D) shows that the bio-3D printed artificial skin cultured in vitro for different times can achieve layered epidermal regeneration. Figures (E) and (F) separately show blood vessel and sebaceous gland regeneration.

[0054] Comparative Example 1:

[0055] Other steps are the same, but during the preparation of the bio-3D printed artificial skin, skin stem cells are not added to the hydrogel material. A hair follicle regeneration model is established for the prepared artificial skin to detect the wound healing and hair follicle regeneration. The test results show that the prepared injectable hydrogel can heal the wound, but hair follicle regeneration cannot be achieved.

[0056] The above are only the preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. For those skilled in the art of this technology, the improvements and transformations obtained without departing from the technical concept of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of a prefabricated bio-3D printed artificial skin for skin and hair follicle regeneration, characterized in that, It includes the following steps: S1: Configure the hydrogel material for the bio-3D printed artificial skin. Each milliliter of the hydrogel material contains 50 mg of methacrylated gelatin (GelMA), 5 mg of methacrylated hyaluronic acid (HAMA), and 5 mg of lithium phenyl-2,4,6-trimethylbenzoylphosphate (LAP) photoinitiator; S2: Debug the bio-3D printing model of the hydrogel material in step S1 to determine the printer parameters; S3: Add skin stem cells to the hydrogel material prepared in step S1, with 10 7 ~10 8 cells per milliliter; S4: Transfer the multi-component hydrogel material prepared in step S3 into a syringe. After pre-cooling in the refrigerator for 3 minutes, transfer it into a bio-3D printer and prepare the bio-3D printed artificial skin using the printing parameters determined in step S2; S5: Perform UV cross-linking molding on the bio-3D printed artificial skin prepared in step S4 and culture it in vitro.

2. The preparation method of a prefabricated bio-3D printed artificial skin for skin and hair follicle regeneration according to claim 1, characterized in that, In step S2, the printer parameters are: nozzle aperture 0.45 mm, nozzle extrusion distance 0.1 mm, layer-by-layer printing height 0.15 mm, and nozzle movement speed 8 mm / s.

3. The preparation method of a prefabricated bio 3D printed artificial skin for skin and hair follicle regeneration according to claim 1, characterized in that, In step S3, the skin stem cells include epidermal stem cells and dermal stem cells, and the number content ratio of epidermal stem cells to dermal stem cells is 1:

2.

4. The preparation method of a prefabricated bio-3D printed artificial skin for skin and hair follicle regeneration according to claim 1, characterized in that, In step S5, the specific steps are as follows: First, perform ultraviolet light irradiation treatment on the bio-3D printed artificial skin, with the distance from the ultraviolet lamp being 10 - 20 cm and the irradiation time being 150 - 300 s.

5. The preparation method of a prefabricated bio-3D printed artificial skin for skin and hair follicle regeneration according to claim 1, characterized in that, After the artificial skin is cured in step S5, transfer it into a CnT-Prime 3D Barrier special medium and place it in a 37°C constant temperature incubator for in vitro culture.

6. The preparation method of the skin stem cells according to claim 3, characterized in that, The epidermal stem cells are obtained by shearing and separating the skin tissue, digesting with protein disperse enzyme and collagenase, screening and collecting cells, and finally performing adherent culture using Keratinocyte-SFM epidermal keratinocyte medium.

7. The method for preparing skin stem cells according to claim 4, wherein The dermal stem cells are obtained by shearing and separating the skin tissue, digesting with protein disperse enzyme and collagenase, screening and collecting cells, and finally culturing in a DMEM / F12 medium containing B27, EGF, and bFGF.

8. A method for preparing a prefabricated bio-3D printed artificial skin for skin and hair follicle regeneration as described in claim 1 can be used to promote wound skin and hair follicle regeneration.

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