CN111686309A

CN111686309A - Preparation method of 3D printed skin

Info

Publication number: CN111686309A
Application number: CN202010521539.7A
Authority: CN
Inventors: 苏健强; 钟金淑子
Original assignee: Print Rite Unicorn Image Products Co Ltd
Current assignee: Print Rite Unicorn Image Products Co Ltd
Priority date: 2019-04-09
Filing date: 2019-04-09
Publication date: 2020-09-22
Also published as: CN109876197A; CN109876197B

Abstract

The invention provides a preparation method of 3D printed skin, wherein the 3D printed skin comprises a skin layer made by 3D printing, and the 3D printed skin also comprises microneedles; printing the substrate using a 3D printer; printing a skin layer and a plurality of microneedles layer by layer; or printing skin layers layer by layer, and then inserting a plurality of prepared microneedles; or placing a plurality of prepared microneedles, and printing skin layers layer by layer; or the microneedles are printed while the skin layer is printed layer by layer, and the preparation method can prepare the 3D printed skin which is completely attached to the damaged part of the human skin and has a substance conveying function.

Description

Preparation method of 3D printed skin

Technical Field

The invention relates to the field of 3D printing and tissue engineering biomedical materials, in particular to a preparation method of 3D printed skin.

Background

The skin is the largest organ of the human body, and not only has the function of resisting external environmental infection, but also plays a role in preventing water, electrolytes and other substances in the body from being lost. In general, skin can recover itself if it is mildly damaged. When a large area of skin is severely damaged, such as a burn, the doctor must immediately introduce fluid and protect the wound. If only the superficial layer of skin is damaged, new skin may regenerate in a normal healthy person, but the skin repair process is very difficult in some patients, such as diabetics. Furthermore, if the patient is subjected to a severe burn, affecting the endogenous stem cells, the skin cannot be repaired by itself, and superficial skin from other parts of the body must usually be transplanted to the wound, but this method causes new scarring. If the burn is large in area, the skin of the human body is almost remained, and the transplantation cannot be carried out. Without skin protection, severe burns can have severe dehydration and bacterial infections that can be life threatening, in which case artificial skin is needed.

Artificial skin is a skin substitute artificially developed in vitro by using the principles and methods of bioengineering and regenerative biology to repair and replace defective skin tissues. The artificial skin is artificially synthesized, so that the survival rate of the severe burn patient can be greatly improved. The variety of artificial skin is various, and the artificial skin which is researched and applied all over the world at present has the following three types: (1) artificial skin in the form of film or sponge made of synthetic materials such as polymer plastics, synthetic polypeptide, and artificial fiber; (2) artificial skin made of regenerated protein and animal tissue such as amnion, peritoneum, and fetal membrane; (3) sheet-like artificial skin made of fully polymerized film.

With further maturation of the technology, it has become possible to cultivate full-thickness skin having the same properties as human skin. However, most of the current artificial skins are printed manually or on a flat surface, and the production efficiency and the treatment effect are limited. Prior patent application documents disclose 3D printed skin and methods for making the same. However, in clinical treatment, in order to accelerate wound healing and promote the growth of body cells, the skin wound needs to be kept in a certain moist state, i.e. the wound is required to be neither excessively dry nor excessively moist. The artificial skin prepared by the existing method has the defects of different aspects and different degrees in the treatment process, or has poor strength due to poor adhesion; or the antigenicity is too strong, and the irritation is large; or the permeability is poor, so the fabric is easy to infect; or scar accumulation due to the inhibition of the growth of autogenous skin; or the raw materials are not easy to be obtained, so the cost is too high; or the preparation and storage are difficult due to complicated process, so that the method cannot be well popularized and clinically applied.

Disclosure of Invention

In order to solve the above problems, it is a primary object of the present invention to provide a method for preparing 3D printed skin that is completely attached to a damaged portion of human skin and has a substance transfer function.

In order to achieve the main purpose of the invention, the invention provides a preparation method of 3D printed skin, the 3D printed skin comprises a skin layer made by 3D printing, wherein the 3D printed skin also comprises a microneedle, the preparation method comprises the steps of scanning a damaged part of the skin by using a 3D scanner, acquiring the attribute of the skin required by the damaged part, establishing a substrate model and a 3D printed skin model; printing the substrate using a 3D printer; printing a skin layer and a plurality of microneedles layer by layer; or printing skin layers layer by layer, and then inserting a plurality of prepared microneedles; or placing a plurality of prepared microneedles, and printing skin layers layer by layer; alternatively, the microneedles are printed at the same time as the skin layer is printed layer by layer.

Therefore, the skin tissue with the specific thickness and the specific shape at the damaged position of the human skin is printed by adopting the 3D scanning modeling technology and the 3D layered printing and layer-by-layer stacking method, and the full-customized skin can be printed according to actual needs. The microneedles can be formed by printing while the biological 3D layer-by-layer skin layer is printed, or the microneedles are implanted for a second time after the 3D layer is printed. The skin layer can be printed layer by layer from inside to outside or from outside to inside when the skin layer is printed layer by layer. In addition, the microneedles may be constructed using various materials as desired. The materials of construction may be pharmaceutical grade stainless steel or other metals, metal alloys, silicon dioxide, polymeric materials, composite materials, and the like. The polymer material may be natural polymer material or synthetic polymer. Representative biodegradable polymers include polymers of hydroxy acids such as lactic acid and glycolic acid, such as polylactide, polyglycolide, and polylactide-co-glycolide, and the like, and the biodegradable polymers can also be copolymers copolymerized with polyethylene glycol, polyanhydrides, polyorthoesters, polyurethanes, poly (butyric acid), poly (valeric acid), and poly (lactide-co-caprolactone), and the like. Representative non-biodegradable polymers include polycarbonates, polyesters, polyacrylamides, and the like. The presence of microneedles can cause pain to the patient when the nerve grows to the epidermis, and thus the microneedles should be removed or degenerated when the skin is repaired and the nerve grows back. When non-biodegradable materials are used, the microneedles should have sufficient mechanical strength so that the microneedles can remain intact when inserted into the biological barrier, when held in place for many days, and when removed. When a biodegradable material is employed, the microneedle must remain intact for a sufficient period of time to enable the microneedle to serve its intended purpose, e.g., the purpose of the microneedle being used as a catheter for delivering a drug. The microneedles are preferably made of biodegradable material, and the microneedles degrade after regeneration after implantation into the skin, and help maintain the barrier between the epidermis and the underlying connective tissue.

Preferably, the step of obtaining the desired skin properties of the damaged area comprises: a three-dimensional model and a skin texture model of the printed substrate are obtained.

Another preferred solution is that the established 3D printed skin model comprises a scaffold; the bracket is arranged above the surface of the skin layer, below the bottom surface of the skin layer or in the skin layer; the stent is made of biodegradable material or non-biodegradable material.

From the above, the stent is beneficial to fixation of implanted skin and is convenient for surgical suture. The stent may be a continuous whole, may have a gap in the middle, or may be divided into multiple stents, as long as it can support in the skin layer. Preferably, the scaffold is made of a biodegradable and absorbable biopolymer nanofiber material. The scaffold can be degraded or removed after 3D printed skin grafting.

Another preferred embodiment is to determine whether stem cells are needed before the step of printing the substrate using the 3D printer, and if so, obtain a small amount of skin tissue, isolate primary skin cells, and culture to obtain the required amount of dermal and epidermal cells for printing the skin layer.

Further, the step of isolating the primary skin cells comprises: cleaning, disinfecting, weighing and chopping skin tissues to obtain primarily treated skin tissues, and adding corresponding protease and collagenase to carry out tissue enzymolysis to obtain free skin cell tissue fluid; and filtering skin cell tissue fluid, centrifuging to gather cells, and inoculating the cells to a culture dish to obtain primary skin cells.

In another preferred embodiment, the step of culturing the dermal cells and epidermal cells to obtain the desired amount comprises: performing primary culture, namely obtaining a part of primary cells, adding a ROCK inhibitor for culture, and performing subculture for a period of time to obtain the required quantity of epidermal stem cells; and (3) obtaining a part of primary cells, culturing without adding a ROCK inhibitor, and subculturing for a period of time to culture the dermal stem cells with the required amount.

From the above, 3D printed skin including a dermal cell layer and an epidermal cell layer can be constructed close to human skin, and can generate a horny layer and hair. The cell layer may carry antibiotics such as minocycline. When this skin configuration is employed, the second ends of the microneedles reach at least the dermal layer of the 3D printed skin to satisfy delivery of the nutrients or agents. The collagen gel material may also be replaced by other biodegradable materials or mixtures with other biodegradable materials. The 3D printed skin can also be a compact film layer, and is formed by mixing, melting and depositing polycarboxylic anhydride and polyethylene glycol succinate for 3D printing, and collagen, polylysine, glycine, Arabic gum, citric acid, drugs with antibacterial and antiviral effects and the like are filled and input through the micro-needle after being implanted, so that the manufacturing difficulty of the whole skin can be further reduced.

In another preferred embodiment, the substrate is a polydimethylsiloxane substrate.

Another preferred embodiment is where the microneedles are embedded in the skin layer, with a first end of the microneedles penetrating to or through the surface of the skin layer and a second end of the microneedles near, penetrating to or through the bottom surface of the skin layer; the microneedle comprises at least one passageway disposed between a first end and a second end.

From top to bottom, 3D prints skin and implants the back, and the micropin second end can be close to, contact or pierce the acceptor end, and first end is attached in the outside on skin layer, can realize that nutrient solution, medicine or cell from 3D prints skin outward to inside transport, and the micropin can also realize excrement etc. and carry to implanting skin outward from implanting the internal cell of person simultaneously to be favorable to implanting the regeneration of skin.

Further, the microneedle is provided with at least one through hole communicated with the passage.

From the above, the micro-needle can be provided with a plurality of through holes to realize the transportation in different modes. For example, through holes are formed on the side surfaces of the microneedles, so that the positioning input or the excrement output of the side surfaces is realized. In addition, the use of microneedles as catheters can serve the purpose of delivering drugs, for example, by infusing drugs, nutrient solutions, or cells through the microneedles. The agent may include minocycline to prevent infection and maintain the cells. The medicament can also comprise traditional Chinese medicine extracts and the like. The nutrient solution may include growth factors, such as factors that stimulate nerve growth, for stimulating nerve growth to the epidermis, so that the implanted skin can protect itself in response to touch, pain, stretching, pressure, etc., and additionally, can promote hair growth. The infused cells may also include mesenchymal stem cells with minocycline, and the like. Furthermore, the first end of the microneedle is connected to a reservoir for holding a medicament, nutrient solution or cells, and the reservoir is connected to the first end of the microneedle, so that the medicament, nutrient solution or cells stored or generated in the reservoir can flow out of the reservoir, pass through the passage of the microneedle and flow into the target tissue. When attached to the reservoir, it is preferred that the first ends of the microneedles protrude through the surface of the skin. In addition, a sensor may be used to release the substance of the reservoir. In addition, because the artificial skin can be transplanted successfully or not, the key point is whether rapid vascularization can be realized, and therefore, the nutrition should be supplied as soon as possible after the skin is transplanted. The nutrient solution input by the invention can also comprise a factor (VEGF) gene capable of promoting vascularization, and the VEGF gene is successfully transferred into human fiber cells to enable the human fiber cells to secrete VEGF so as to promote vascularization. The invention can also implant autologous endothelial cells and fibroblasts into the dermal scaffold through a proper way to induce the formation of new blood vessels. In clinical application, for example, in a sieve-shaped skin grafting method, a plurality of small openings are cut in an artificial skin to be subjected to skin grafting, the artificial skin is fixed on a wound surface under a certain tension condition, so that the area of a skin piece can be increased, and meanwhile, the drainage of exudate is facilitated.

In a further scheme, a plurality of prepared microneedles are subjected to sterilization treatment; the printing of the skin layer is performed in a clean closed environment.

Therefore, the 3D printed skin provided by the invention is implanted on the surface of a human body, and the 3D printed skin is ensured to be clean as much as possible, so that infection is avoided. Microneedles of the present invention can be sterilized using standard methods, such as ethylene oxide sterilization or gamma radiation sterilization.

Drawings

Fig. 1 is a schematic structural diagram of a first embodiment of a 3D printed skin according to the present invention, wherein (a) is a schematic diagram of a 3D printed skin; (b) is a schematic view of a microneedle-bearing stent; (c) a partial cross-sectional enlarged view of the skin is printed for 3D.

FIG. 2 is a schematic structural diagram of a second embodiment of 3D printed skin according to the present invention, wherein (a) is a schematic diagram of 3D printed skin; (b) is a schematic view of a microneedle-bearing stent; (c) a partial cross-sectional enlarged view of the skin is printed for 3D.

Fig. 3 is a schematic structural diagram of a third embodiment of 3D printed skin according to the present invention, wherein (a) is a schematic diagram of 3D printed skin; (b) is a schematic view of a microneedle-bearing stent; (c) a partial cross-sectional enlarged view of the skin is printed for 3D.

Fig. 4 is a schematic structural diagram of a fourth embodiment of the 3D printed skin of the present invention, wherein (a) is a schematic diagram of the 3D printed skin; (b) is a schematic view of a microneedle-bearing stent; (c) a partial cross-sectional enlarged view of the skin is printed for 3D.

FIG. 5 is a schematic diagram of a fifth embodiment of 3D printed skin of the present invention, wherein (a) is a schematic diagram of 3D printed skin; (b) an exploded schematic view of a 3D printed skin.

Fig. 6 is a partial cross-sectional view of a six-graft embodiment of 3D printed skin according to the present invention.

Fig. 7 is a partial cross-sectional view of a 3D printed skin embodiment of the present invention after implantation.

Fig. 8 is a partial cross-sectional view of an eight-graft embodiment of 3D printed skin according to the present invention.

The invention is further explained with reference to the drawings and the embodiments.

Detailed Description

Embodiment of 3D printing skin preparation method

The embodiment provides a preparation method of a 3D printed skin, which includes the following steps performed in sequence:

(1)3D scanning and skin modeling: scanning the damaged part of the skin of a human body by using a 3D scanner to obtain a three-dimensional structure of the damaged part of the skin, calculating various attributes of the full-custom skin to be transplanted to the wound by using software, including various attributes of a base three-dimensional model and a skin structure model, and establishing a 3D printing skin model which is in accordance with the three-dimensional structure of the damaged part of the skin and comprises a bracket, so that the custom skin is completely attached to the wound; estimating and evaluating whether stem cells are needed, and if so, primarily estimating the quantity of the stem cells; meanwhile, the type of nutrient solution or medicament needing to be input for promoting skin regeneration is evaluated, the structure of the microneedle, the position and the layout of the microneedle, the sizes of the microneedle passage and the through hole and the like are evaluated.

(2) When stem cells are needed, stem cells are prepared: extracting a small amount of human skin tissue, cleaning, sterilizing, weighing, chopping to obtain primarily treated skin tissue, adding corresponding protease and collagenase, and performing tissue enzymolysis to obtain free skin cell tissue fluid; filtering to obtain cell sap, centrifuging to gather cells, and inoculating the cells to a culture dish to obtain primary skin cells; performing primary culture, namely adding a ROCK inhibitor into a part of primary cells for culture, and performing subculture for a period of time to obtain the required quantity of epidermal stem cells; and (3) taking a part of primary cells, culturing without adding a ROCK inhibitor, and subculturing for a period of time to culture the dermal stem cells with the required amount. The stem cells can be dermal stem cells and endogenous epidermal stem cells. The obtained epidermal stem cells and dermal stem cells can be stored in liquid nitrogen for printing, and when printing is needed, the cells are transferred to a raw material box of a printer.

(3)3D layered printing: printing out Polydimethylsiloxane (PDMS) substrate through the 3D printer firstly, printing a collagen gel layer on the PDMS substrate, printing a dermis cell layer on the collagen gel layer, printing the collagen gel layer on the dermis cell layer, printing a epidermis cell layer on the collagen gel layer, and printing the collagen gel layer on the epidermis cell layer. The printing process of each layer of collagen gel is as follows: printing a layer of cross-linking agent, printing a layer of collagen gel on the cross-linking agent, and then printing the cross-linking agent to fix the layer. The printing process of the cell layer is as follows: printing a layer of cross-linking agent, printing a layer of collagen gel on the cross-linking agent, printing a layer of cell layer on the collagen gel, and then printing the cross-linking agent to fix the collagen gel layer. And the number and the thickness of the layer of the eudermal cell layer, the collagen gel layer and the epidermal cell layer are determined by modeling calculation according to actual needs. In another embodiment of the method of the present invention, the printing may also be from the skin surface to the subcutaneous layer. The microneedles can be formed by printing while the biological 3D layer-by-layer skin layer is printed, or the microneedles are implanted for a second time after the 3D layer is printed.

After the 3D printing skin is prepared, skin tissue culture and transplantation are carried out: and transferring the printed skin into a culture dish, culturing for 2-10 days at a gas-liquid interface of a constant temperature box, and transplanting the skin tissue to the damaged part of the skin or performing an in-vitro skin model test after the skin tissue is primarily fused.

According to the 3D skin preparation method provided by the embodiment, the skin is divided into the dermal cell layer, the epidermal cell layer and the gel layer according to modeling, the full-custom skin close to the structure of the human skin is printed in a multi-layer printing and layer-by-layer stacking mode, the full-function skin can generate the cuticle and the hair, and nutrient solution or medicament can be delivered through the micro-needle arranged on the skin in the implantation process.

3D printing skin embodiments

The first embodiment is as follows: as shown in fig. 1, the present embodiment provides a 3D-printed skin including an epidermal cell layer 11, a dermal cell layer 12, microneedles 13, and a scaffold 14. Wherein, the first end of the micro-needle 13 penetrates the surface of the epidermal cell layer 11, the second end penetrates the bottom surface of the dermal cell layer 12, and a passage is arranged in the middle of the micro-needle 13. The microneedles 13 include straight sections and tapered sections. The microneedles 13 are made of the same material as the holder 14. The stent 14 is located on the uppermost layer, and the stent 14 can be removed after 3D printing skin graft surgery. The 3D printed skin may also comprise a layer of collagen gel, not shown in the figures.

Example two: as shown in fig. 2, the present embodiment provides a 3D-printed skin including an epidermal cell layer 21, an endothelial cell layer 22, microneedles 23, and a scaffold 24. Wherein, the micro-needle 23 and the bracket 24 are made of different materials. The stent 24 is positioned on the topmost layer of the 3D printed skin and can be removed after the 3D printed skin graft surgery. The rest of the structure is similar to the first embodiment.

Example three: as shown in fig. 3, the present embodiment provides a 3D-printed skin including an epidermal cell layer 31, an endothelial cell layer 32, microneedles 33, and a scaffold 34. Among other things, the scaffold 34 is located in a middle layer of the 3D printed skin, which can degrade after the 3D printed skin graft surgery. The microneedles 33 and the stent 34 are made of the same or different materials. The rest of the structure is similar to the first embodiment.

Example four: as shown in fig. 4, the present embodiment provides a 3D-printed skin including an epidermal cell layer 41, an endothelial cell layer 42, microneedles 43, and a scaffold 44. Where the scaffold 44 is located at the lowest layer of 3D printed skin, it may degrade after 3D printed skin grafting surgery. The microneedles 43 and the holders 44 are made of the same or different materials. The rest of the structure is similar to the first embodiment.

Example five: as shown in fig. 5, the present embodiment provides a 3D-printed skin including an epidermal cell layer 51, a dermal cell layer 52, microneedles 53, and a scaffold 54. Wherein the microneedles 53 are tapered. The scaffolds 54 are positioned on the topmost layer of the 3D printed skin, dispersed around the microneedles 53, which may be removed after the 3D printed skin grafting procedure. The microneedles 53 are made of the same or different materials as the stent 54.

Example six: as shown in fig. 6, the present embodiment provides a 3D-printed skin including an epidermal cell layer 61, an endothelial cell layer 62, microneedles 63, and a scaffold 64. Wherein a passage 65 is arranged between the first end and the second end of the microneedle 63, the microneedle 63 is tapered, and the diameter of the microneedle 63 is gradually reduced from the first end to the second end. The microneedles 63 are also provided with through-holes 66 at the sides thereof. After the 3D printed skin is transplanted, the second end of the microneedle 63 is inserted into a patient body 67, nutrient solution, medicament or cells 68 can be input from the first end of the microneedle 63 through the passage 65, the nutrient solution, medicament or cells 68 can be positioned, permeated or conveyed to the patient from the second end of the microneedle 63 or the through hole 66, in addition, waste can be discharged out of the skin through the passage 65, and the regeneration, fusion and growth of the skin are facilitated. A scaffold 64 is disposed around the microneedles 63 and in the epidermal cell layer 61 and the dermal cell layer 62, and is degradable after 3D printing of the skin graft.

Example seven: as shown in fig. 7, the present embodiment provides a 3D-printed skin including an epidermal cell layer 71, an endothelial cell layer 72, microneedles 73, and a scaffold 74. The micro-needle 73 is provided with a passage 75 and a through hole 76, the second end of the micro-needle 73 is inserted into the body 77 of a patient, and the micro-needle 73 can deliver nutrient solution, medicament or cells 78 and also can discharge waste. A scaffold 74 is disposed apart from the microneedles 73, which are located in the epidermal cell layer 71 and the dermal cell layer 72, and is degradable after 3D printing of the skin graft. The rest of the structure is similar to the sixth embodiment.

Example eight: as shown in fig. 8, the present embodiment provides a 3D-printed skin including an epidermal cell layer 81, an endothelial cell layer 82, microneedles 83, and a scaffold 84. The micro-needle 83 is provided with a passage 85 and a through hole 86, the second end of the micro-needle 83 is inserted into a patient body 87, and the micro-needle 83 can deliver nutrient solution, medicament or cells 88 and also can discharge waste. In the embodiment, the diameter of a part of the micro-needle 83 is gradually reduced from the first end to the second end, and the diameter of a part of the micro-needle 83 is gradually reduced from the second end to the first end, so that the micro-needle density is improved, the substance is conveyed, and the micro-needle can automatically and rapidly heal with the skin of a human body.

The 3D printed skin provided by the invention can be used for repairing the skin of patients with various skin diseases such as psoriasis, acne, albinism and the like, and can also be used for repairing the skin of patients with burn, diabetes and the like. And 3D printing skin is full customization skin, and is identical with the wound completely, has improved the treatment success rate to can obtain better cure effect. Meanwhile, the microneedle plays an important role in inputting nutrient solution, cells and medicines and discharging waste after 3D printing skin implantation.

Finally, it should be emphasized that the above-described preferred embodiments of the present invention are merely examples of implementations, rather than limitations, and that many variations and modifications of the invention are possible to those skilled in the art, without departing from the spirit and scope of the invention.

Claims

1. A preparation method of 3D printed skin, the 3D printed skin comprises a skin layer made by 3D printing, and is characterized in that:

the 3D printed skin further comprises microneedles, and the method of making comprises:

scanning a damaged part of skin by using a 3D scanner, acquiring the attribute of the skin required by the damaged part, and establishing a base model and a 3D printing skin model;

printing the substrate using a 3D printer;

printing the skin layer and the plurality of microneedles layer-by-layer; or, printing the skin layer by layer, and then inserting the prepared microneedles; or placing the prepared microneedles, and printing the skin layer by layer; alternatively, the microneedles are printed at the same time as the skin layer is printed layer by layer.

2. The method of claim 1, wherein:

the step of obtaining the skin properties required for the damaged area comprises:

a three-dimensional model and a skin texture model of the printed substrate are obtained.

3. The method of claim 1, wherein:

the established 3D printing skin model comprises a bracket;

the scaffold is disposed above the surface of the skin layer, below the bottom surface of the skin layer, or in the skin layer;

the stent is made of biodegradable material or non-biodegradable material.

4. The method of claim 1, wherein:

before the step of printing the substrate by using the 3D printer, judging whether stem cells are needed, if so, obtaining a small amount of skin tissue, separating to obtain primary skin cells, and culturing to obtain the needed amount of dermal cells and epidermal cells for printing the skin layer.

5. The method of claim 4, wherein:

the step of isolating primary skin cells comprises:

cleaning, disinfecting, weighing and chopping the skin tissue to obtain preliminarily processed skin tissue, and adding corresponding protease and collagenase to carry out tissue enzymolysis to obtain free skin cell tissue fluid;

and filtering the skin cell tissue fluid, centrifuging the aggregated cells, and inoculating the cells to a culture dish to obtain the primary skin cells.

6. The method of claim 4, wherein:

the step of culturing to obtain the required amount of dermal cells and epidermal cells comprises:

performing primary culture, namely obtaining a part of primary cells, adding a ROCK inhibitor for culture, and performing subculture for a period of time to obtain the required quantity of epidermal stem cells;

and (3) obtaining a part of primary cells, culturing without adding a ROCK inhibitor, and subculturing for a period of time to culture the dermal stem cells with the required amount.

7. The method of claim 1, wherein:

the substrate is a polydimethylsiloxane substrate.

8. The method of claim 1, wherein:

the microneedles are embedded in the skin layer, a first end of the microneedles is threaded to or through a surface of the skin layer, and a second end of the microneedles is near, threaded to or through a bottom surface of the skin layer;

the microneedle comprises at least one passageway disposed between the first end and the second end.

9. The method of claim 8, wherein:

the microneedle is also provided with at least one through hole communicating with the passageway.

10. The production method according to any one of claims 1 to 9, characterized in that:

sterilizing the prepared microneedles;

the printing of the skin layer is performed in a clean closed environment.