Imitated skin ridge microneedle loaded with epidermal stem cells as well as preparation method and application of imitated skin ridge microneedle

Abstract

The invention relates to the field of tissue engineering biological materials, in particular to an epidermal stem cell-loaded imitation skin ridge microneedle and a preparation method and application thereof, and the method comprises the following steps: providing a target skin sample; a three-dimensional structure of the target skin sample is obtained through laser femtosecond three-dimensional reconstruction, image processing software is adopted for analysis and quantification, and skin ridge size parameters of the target skin sample are obtained; preparing a microneedle mold according to the skin ridge size parameters; respectively preparing a needle tip solution and a substrate solution, and adding the epidermal stem cells in the culture logarithmic phase into the needle tip solution to obtain a mixed solution; filling a microneedle area of a microneedle mold with the mixed solution, and performing centrifugal treatment; and filling a substrate area of a microneedle mold with the substrate solution, carrying out illumination crosslinking, and demolding to obtain the epidermal stem cell-loaded artificial skin ridge microneedle. The method can realize stable survival of epidermal stem cells in the artificial skin ridge microneedle, and has the effects of reconstructing a skin wound skin ridge structure and improving wound healing quality.

Description

Technical Field

[0001] The present invention relates to the field of tissue engineering biomaterials, and particularly to a dermal ridge-like microneedle loaded with epidermal stem cells, a preparation method thereof, and an application thereof. Background Art

[0002] Traditional skin grafting treatment depends on the patient having sufficient skin sources. Although skin grafting can quickly close the wound, it causes great iatrogenic damage to the skin grafting site, and problems such as scar hyperplasia and intolerance to friction often accompany wound repair. The transplantation of tissue-engineered skin substitutes (such as tissue-engineered composite skin constructed by autologous epidermal cell sheets, cells and scaffold materials) can solve the problem of lack of skin sources in patients. However, the in vitro preparation period of ESS (Engineered Skin Substitute) is long, and it lacks the characteristic undulating microstructure (called dermal ridges) at the dermal-epidermal junction, resulting in poor mechanical properties of the newly formed skin, easy ulceration, blister formation and other quality problems.

[0003] Through histopathological observation, it is found that compared with normal skin, the lack of dermal ridges is a particularly significant feature in the skin after the healing of large-area wounds treated by current surgical methods. In human skin, minor wounds can heal effectively without treatment. However, it is difficult to regenerate dermal ridges in wounds with large-area skin defects such as burns. Dermal ridges are specific niches for epidermal stem cells and play an important role in regulating stem cell development, maintaining skin function homeostasis, and improving the quality of wound repair. Therefore, in situ reconstruction of the dermal ridge structure in burn wounds is the key to solving the problem of poor wound repair quality. Summary of the Invention

[0004] The purpose of the present invention is to provide a dermal ridge-like microneedle loaded with epidermal stem cells, a preparation method thereof, and an application thereof, which can achieve the stable survival of epidermal stem cells in the dermal ridge-like microneedle, and the prepared dermal ridge-like microneedle loaded with epidermal stem cells has the function of reconstructing the dermal ridge structure of skin wounds and improving the quality of wound healing.

[0005] In order to achieve the above purpose, the technical scheme adopted by the present invention is as follows:

[0006] In the first aspect, the present invention provides a preparation method of a dermal ridge-like microneedle loaded with epidermal stem cells, which includes:

[0007] Providing a target skin sample;

[0008] Obtaining the three-dimensional structure of the target skin sample through femtosecond laser three-dimensional reconstruction, analyzing and quantifying the three-dimensional structure by using image processing software, and obtaining the dermal ridge size parameters of the target skin sample;

[0009] Preparing a microneedle mold according to the dermal ridge size parameters;

[0010] Prepare the tip solution and the substrate solution respectively, add epidermal stem cells in the logarithmic growth phase to the tip solution to obtain a mixed solution;

[0011] Fill the mixed solution into the microneedle area of the microneedle mold and perform centrifugation; then fill the substrate solution into the substrate area of the microneedle mold, perform photo-crosslinking, and demold to obtain microneedles with epidermal stem cells loaded on the skin ridge-like microneedles.

[0012] Furthermore, the preparation of the epidermal stem cells in the logarithmic growth phase includes:

[0013] Take the target skin sample, digest and separate it, remove the dermal tissue, and retain the epidermal tissue;

[0014] Crush, digest, filter, and centrifuge the epidermal tissue to collect cells;

[0015] Adjust the cell concentration to (1 - 2)×10 6 cells / 25 cm 2 with Epilife culture medium, inoculate it into a culture flask coated with type IV collagen, and culture to obtain epidermal stem cells.

[0016] Furthermore, the concentration of epidermal stem cells in the logarithmic growth phase in the mixed solution is (0.2 - 0.5)×10 7 / mL.

[0017] Furthermore, the rotation speed of the centrifugation is set to 800 - 1500 rpm, and the centrifugation time is set to 2 - 7 min.

[0018] Furthermore, by weight percentage, the tip solution includes 0.1 - 0.25% of LAP, 5 - 15% of GelMA, 5 - 15% of HAMA, and 69.75 - 89.9% of the culture medium;

[0019] By weight percentage, the substrate solution includes 0.1 - 0.25% of LAP, 5 - 15% of GelMA, and 84.75 - 94.9% of the culture medium.

[0020] Furthermore, it also includes: after obtaining the skin ridge-like microneedles with epidermal stem cells loaded, place the skin ridge-like microneedles with epidermal stem cells loaded in the culture medium, and culture for 24 - 72 hours under the conditions of a temperature of 37°C and a carbon dioxide concentration of 5%.

[0021] In a second aspect, the present invention provides a skin ridge-like microneedle with epidermal stem cells loaded, which is prepared by the above-mentioned preparation method of the skin ridge-like microneedle with epidermal stem cells loaded, and includes a substrate, a tip part connected to the substrate, and epidermal stem cells loaded on the tip part.

[0022] In a third aspect, the present invention provides an application of the above-mentioned skin ridge mimicking microneedles loaded with epidermal stem cells in a skin wound repair device.

[0023] The present invention has the following unexpected beneficial effects:

[0024] 1. The present invention obtains the three-dimensional structure of a target skin sample through femtosecond laser three-dimensional reconstruction and analyzes and quantifies it to obtain skin ridge size parameters, enabling precise simulation of the characteristic structure of skin ridges. This precise simulation provides a basis for preparing microneedles with bionic characteristics, making the skin ridge mimicking microneedles closer to real skin in structure, helping to restore the normal physiological functions of the skin, improving the quality of wound healing, promoting better wound healing, reducing adverse consequences such as scar formation, and being of great significance for skin injury repair.

[0025] 2. The present invention prepares bionic skin ridge mimicking microneedles from materials such as GelMA and HAMA, and prepares cell-loaded skin ridge mimicking microneedles by reasonably limiting the epidermal stem cell density and the concentrations of GelMA and HAMA, achieving stable cell survival in the microneedles. GelMA (glycidyl methacrylate gelatin) and HAMA (2-hydroxyethyl methacrylate hyaluronic acid) are materials with good biocompatibility. Preparing bionic skin ridge mimicking microneedles from them can endow the microneedles with appropriate biodegradability, mechanical properties and biological activity. Reasonably limiting the epidermal stem cell density and the concentrations of GelMA and HAMA is crucial for the stable survival of cells in the microneedles. An appropriate cell density can avoid the influence of cell overcrowding or sparsity on cell growth and function, ensuring normal communication and cooperation between cells. Precise control of the concentrations of GelMA and HAMA can create an ideal microenvironment for cells, achieving stable cell survival in the microneedles. The stably surviving epidermal stem cells can continuously play a role at the wound site, such as differentiating into various skin cells, participating in the repair and regeneration of skin tissue, promoting the reconstruction of skin ridge structure, thereby improving the quality of wound healing, reducing the occurrence of complications, and ultimately achieving better treatment effects. Description of the Drawings

[0026] Figure 1 Shows a flowchart of the preparation method of the skin ridge mimicking microneedles loaded with epidermal stem cells according to an embodiment of the present invention.

[0027] Figure 2 Shows a schematic structural diagram of the skin ridge mimicking microneedles loaded with epidermal stem cells prepared according to an embodiment of the present invention.

[0028] Figure 3 Shows a schematic diagram of the characteristic structure of skin ridges simulated and constructed by femtosecond laser three-dimensional technology.

[0029] Figure 4The figure shows the schematic diagram of different layers of the skin ridge - like microneedles loaded with epidermal stem cells prepared by the present invention under a microscope after calcein - AM staining.

[0030] Figure 5 The figure shows the macroscopic schematic diagram of the wound repair conditions of the control group, comparison group 1, comparison group 2, and experimental group.

[0031] Figure 6 The figure shows the schematic diagram of the wound healing rates of the control group, comparison group 1, comparison group 2, and experimental group.

[0032] Figure 7 The figure shows the schematic diagram of the regeneration of the skin ridge structure after wound repair in the control group, comparison group 1, comparison group 2, and experimental group. Detailed implementation manners

[0033] The following will describe the implementation manners of the present invention with reference to the accompanying drawings and preferred embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for explaining the present invention and not for limiting the protection scope of the present invention.

[0034] In one embodiment, the present invention provides a method for preparing skin ridge - like microneedles loaded with epidermal stem cells, including:

[0035] Providing a target skin sample;

[0036] Obtaining the three - dimensional structure of the target skin sample through femtosecond laser three - dimensional reconstruction, analyzing and quantifying the three - dimensional structure using image - processing software to obtain the skin ridge size parameters of the target skin sample;

[0037] Preparing a microneedle mold according to the skin ridge size parameters;

[0038] Respectively preparing a tip solution and a substrate solution, adding epidermal stem cells in the logarithmic growth phase to the tip solution to obtain a mixed solution;

[0039] Filling the mixed solution into the microneedle area of the microneedle mold and performing centrifugation; then filling the substrate solution into the substrate area of the microneedle mold, cross - linking by light, and demolding to obtain the skin ridge - like microneedles loaded with epidermal stem cells.

[0040] The present invention obtains the three-dimensional structure of a target skin sample through femtosecond laser three-dimensional reconstruction, analyzes and quantifies it to obtain the dermal ridge size parameters, and can accurately simulate the characteristic structure of skin dermal ridges. This accurate simulation provides a basis for preparing microneedles with bionic characteristics, making the dermal ridge-mimicking microneedles closer to real skin in structure, helping to restore the normal physiological functions of the skin, improving the quality of wound healing, promoting better wound healing, reducing adverse consequences such as scar formation, and being of great significance for skin injury repair.

[0041] Specifically, as shown in Figure 1 the method for preparing the dermal ridge-mimicking microneedles loaded with epidermal stem cells according to the present invention comprises the following steps:

[0042] S1, providing a target skin sample: providing a reference standard for the subsequent preparation of dermal ridge-mimicking microneedles, ensuring that the microneedles can simulate the characteristics of the target skin to the greatest extent, and laying a foundation for preparing microneedles that match the actual skin structure and function.

[0043] S2, obtaining the three-dimensional structure of the target skin sample through femtosecond laser three-dimensional reconstruction, and analyzing and quantifying the three-dimensional structure using image processing software to obtain the dermal ridge size parameters of the target skin sample.

[0044] Femtosecond laser three-dimensional reconstruction can accurately obtain the three-dimensional structure information of the target skin sample, providing accurate data support for the bionics of the microneedles.

[0045] Using image processing software for analysis and quantification helps to accurately determine the dermal ridge size parameters, enabling the prepared microneedles to highly simulate the dermal ridges of real skin in terms of size and structure, thereby improving the compatibility and conformability of the microneedles with the skin.

[0046] S3, preparing a microneedle mold according to the dermal ridge size parameters, ensuring that the microneedle mold can accurately replicate the dermal ridge structure of the target skin, providing a key mold basis for subsequently preparing microneedles with an accurate dermal ridge-mimicking structure, enabling the microneedles to better adapt to the morphology of the skin surface, and improving the piercing effect and treatment effect of the microneedles.

[0047] S4, separately preparing a tip solution and a substrate solution, and adding epidermal stem cells in the logarithmic growth phase to the tip solution to obtain a mixed solution.

[0048] Separately preparing the tip solution and the substrate solution can accurately adjust the composition and properties of the solutions according to the functions and requirements of different parts of the microneedles to achieve the best performance.

[0049] Using epidermal stem cells in the logarithmic growth phase, at this time the cells have strong proliferative ability and good activity, can better play a role in the microneedles, help to improve the treatment effect of the microneedles and promote the repair of skin tissue.

[0050] S5. Fill the mixed solution into the microneedle area of the microneedle mold and perform centrifugation; then fill the substrate solution into the substrate area of the microneedle mold, perform photo-crosslinking, and demold to obtain dermal ridge microneedles loaded with epidermal stem cells.

[0051] Centrifugation helps to evenly distribute the epidermal stem cells in the mixed solution in the microneedle area and can remove the air bubbles in the solution, improving the quality and performance of the microneedles.

[0052] Photo-crosslinking can quickly solidify and form the substrate solution into a stable microneedle structure without damaging the loaded epidermal stem cells.

[0053] The finally demolded dermal ridge microneedles loaded with epidermal stem cells combine the dual advantages of the dermal ridge structure and the loaded epidermal stem cells. They can not only effectively penetrate the skin but also utilize the repair and regeneration ability of epidermal stem cells to achieve the treatment of skin wounds and the repair of skin tissues.

[0054] As a preferred embodiment of the present invention, the preparation of the epidermal stem cells in the logarithmic growth phase includes:

[0055] Take the target skin sample, digest and separate it, remove the dermal tissue, and retain the epidermal tissue;

[0056] Crush, digest, filter, and centrifuge the epidermal tissue to collect cells;

[0057] Adjust the cell concentration to (1 - 2)×10 6 cells / 25 cm 2 with Epilife culture medium, inoculate it into a culture flask coated with type IV collagen, and culture to obtain epidermal stem cells.

[0058] The present invention accurately obtains epidermal stem cells from the target skin sample. Through operations such as removing the dermal tissue, crushing and digesting, it effectively excludes the interference of other cell types, improves the cell purity, provides a guarantee for culturing high-purity epidermal stem cells with specific functions subsequently, and helps to improve the pertinence and effectiveness of the treatment of epidermal stem cells in the microneedles.

[0059] Using Epilife culture medium provides a suitable nutrient environment for epidermal stem cells, ensuring various nutrients and growth factors required for cell growth and proliferation, and maintaining the normal physiological functions and stemness of the cells. Adjusting the cell concentration to an appropriate range can make the cells evenly distributed in the culture flask, fully utilize the culture space and nutrient resources, facilitate the synchronous growth and proliferation of the cells, and improve the efficiency and quality of cell culture. Moreover, coating the culture flask with type IV collagen simulates the in-vivo extracellular matrix environment, enhances the adhesion of epidermal stem cells to the surface of the culture flask, promotes cell attachment, provides a growth microenvironment similar to that in vivo for the cells, helps maintain the characteristics and functions of epidermal stem cells, enables them to better maintain the ability of self-renewal and differentiation, and thus can more effectively play the role of repairing and regenerating skin tissue after being loaded onto the microneedles.

[0060] The logarithmic-phase epidermal stem cells cultured by this preferred embodiment have strong proliferation ability and good activity. Loading them into the skin-ridge-mimicking microneedles can maximize the therapeutic effect of epidermal stem cells. Combining with the structural advantages of the skin-ridge-mimicking microneedles, it can more effectively promote the repair and regeneration of skin tissue, making the prepared skin-ridge-mimicking microneedles loaded with epidermal stem cells have the function of reconstructing the skin-ridge structure of the skin wound and improving the quality of wound healing.

[0061] As a preferred embodiment of the present invention, the concentration of epidermal stem cells cultured in the logarithmic phase in the mixed solution is (0.2 - 0.5)×10 7 / mL. Within this concentration range, the cells can obtain sufficient nutrients and growth space, which is beneficial to maintaining the activity and normal physiological functions of epidermal stem cells. The cell density is neither too high to cause intense nutrient competition and excessive accumulation of metabolites, which affect the cell state, nor too low to result in insufficient interaction between cells, thus ensuring that the cells can maintain good proliferation and differentiation abilities in the microneedles and better play the role of repairing skin tissue.

[0062] A suitable cell concentration helps to form a uniform cell distribution in the microneedles. After the microneedles are inserted into the wound surface, the epidermal stem cells can be evenly released, improving the effect and stability of microneedle treatment. At the same time, such a concentration can match the material and structure of the microneedles, and will not affect the key properties such as the mechanical properties and biocompatibility of the microneedles due to too many or too few cells, ensuring the reliability and safety of the skin-ridge-mimicking microneedles loaded with epidermal stem cells during use.

[0063] This concentration range can provide a sufficient number of epidermal stem cells. When the microneedles act on the wound surface, there are enough cells participating in the repair and regeneration process of skin tissue, accelerating wound healing, promoting the renewal and regeneration of skin cells, thus improving the efficiency of treating skin trauma or improving skin conditions, reducing the number of treatments and treatment time, and bringing a better treatment experience and effect to patients.

[0064] As a preferred embodiment of the present invention, the rotation speed of the centrifugation treatment is set to 800 - 1500 rpm, and the centrifugation treatment time is set to 2 - 7 min.

[0065] Within this range of rotation speed and time, epidermal stem cells in the mixed solution can be moderately precipitated and distributed in the microneedle area of the microneedle mold. If the rotation speed is too low or the time is too short, epidermal stem cells cannot be fully precipitated into the microneedle area, resulting in uneven distribution or insufficient quantity of epidermal stem cells; if the rotation speed is too high or the time is too long, epidermal stem cells may be excessively squeezed or damaged, affecting cell activity. This setting can ensure that epidermal stem cells are evenly and appropriately filled in the microneedle area, laying a foundation for the subsequent preparation of skin-ridge-mimicking microneedles loaded with epidermal stem cells with stable performance and good therapeutic effects.

[0066] Such centrifugation parameters can achieve effective precipitation and distribution of epidermal stem cells in a short time, improving the efficiency of preparing skin-ridge-mimicking microneedles loaded with epidermal stem cells. At the same time, due to the even distribution and guaranteed activity of epidermal stem cells, the quality of the microneedles is improved, and the consistency between batches is better, which is beneficial for large-scale production and clinical application.

[0067] As a preferred embodiment of the present invention, by weight percentage, the tip solution comprises 0.1 - 0.25% LAP, 5 - 15% GelMA, 5 - 15% HAMA, and 69.75 - 89.9% culture medium.

[0068] LAP (photoinitiator): The content is 0.1 - 0.25%. LAP plays a key role in the photopolymerization reaction. It can initiate the polymerization reaction under the irradiation of light with a specific wavelength, causing GelMA (glycidyl methacrylate gelatin) and HAMA (2-hydroxyethyl methacrylate) to crosslink and form a stable three-dimensional network structure, thereby constructing the tip part of the microneedles. An appropriate amount of LAP ensures that the polymerization reaction can proceed quickly and efficiently, while avoiding problems such as potential cytotoxicity or material property changes caused by excessive initiator.

[0069] GelMA: The content is 5 - 15%. GelMA has good biocompatibility and degradability. It can provide a suitable growth microenvironment for epidermal stem cells, supporting cell adhesion, proliferation, and differentiation. Its three-dimensional network structure can mimic the extracellular matrix, helping to maintain cell morphology and function, and it can gradually degrade in vivo without causing long-term foreign body irritation to tissues.

[0070] HAMA: The content is 5 - 15%. The addition of HAMA can regulate the physical properties of the tip solution, such as hardness, flexibility, and water absorption. It acts synergistically with GelMA to endow the microneedle tip with appropriate mechanical strength, enabling it to smoothly penetrate the skin, while also having a certain degree of flexibility to avoid breakage during use. In addition, HAMA can also improve the hydrophilicity of the material, facilitating cell infiltration and nutrient exchange.

[0071] Culture medium: The content is 69.75 - 89.9%. The culture medium provides the necessary nutrients, growth factors, and buffer environment for epidermal stem cells, maintaining cell viability and normal metabolism. In the tip solution, the culture medium is not only a nutrient source for cells but also affects the crosslinking degree and microstructure of the material, thereby regulating the performance of the microneedles.

[0072] As a preferred embodiment of the present invention, by weight percentage, the substrate solution comprises 0.1 - 0.25% LAP, 5 - 15% GelMA, and 84.75 - 94.9% culture medium.

[0073] LAP: Similar to the tip solution, 0.1 - 0.25% LAP is used to initiate the photopolymerization reaction of the substrate solution, ensuring the rapid and stable formation of the substrate.

[0074] GelMA: The content is 5 - 15%. In the substrate, GelMA also exhibits the advantages of biocompatibility and degradability, providing a stable support framework for the entire microneedle structure. It can form a good bond with the GelMA in the tip part, ensuring the integrity and stability of the overall microneedle structure.

[0075] As a preferred embodiment of the present invention, the method for preparing the skin-ridge-mimicking microneedles loaded with epidermal stem cells further comprises: after obtaining the skin-ridge-mimicking microneedles loaded with epidermal stem cells, placing the skin-ridge-mimicking microneedles loaded with epidermal stem cells in the culture medium and culturing them for 24 - 72 hours under the conditions of a temperature of 37°C and a carbon dioxide concentration of 5%.

[0076] After the preparation of the dermal ridge micro - needles loaded with epidermal stem cells, placing them in a culture medium for cultivation allows the epidermal stem cells to have time to adapt to the new environment inside the micro - needles. A temperature of 37°C simulates the normal body temperature of the human body, and a carbon dioxide concentration of 5% helps maintain the acid - base balance of the culture medium. This provides an environment closer to the in - vivo physiological conditions for epidermal stem cells, which is beneficial to cell survival, metabolism, and functional recovery, and reduces the stress and damage effects that may be caused to cells during the preparation process. A cultivation time of 24 - 72 hours enables epidermal stem cells to continue to proliferate and differentiate in a suitable environment, enhancing cell activity and function. Epidermal stem cells with higher activity can more effectively participate in the wound repair and regeneration processes when subsequently applied to the wound surface, thereby improving the therapeutic effect of the micro - needles on skin trauma or the improvement effect on skin healing.

[0077] In another embodiment, the present invention also provides a dermal ridge micro - needle loaded with epidermal stem cells, which is prepared by using the above - mentioned preparation method of the dermal ridge micro - needle loaded with epidermal stem cells. As shown in Figure 2 The prepared dermal ridge micro - needle loaded with epidermal stem cells includes a substrate 1, a tip part 2 connected to the substrate 1, and epidermal stem cells loaded on the tip part 2.

[0078] In another embodiment, the present invention also provides an application of the above - mentioned dermal ridge micro - needle loaded with epidermal stem cells in skin wound repair instruments.

[0079] The following is an analysis and explanation with specific examples.

[0080] A preparation method of a dermal ridge micro - needle loaded with epidermal stem cells includes:

[0081] S1, providing a target skin sample: separating a thigh skin sample from a clinical patient after surgery, with a length of 1 cm, a width of 1 cm, and a thickness of full - thickness skin.

[0082] S2, obtaining the three - dimensional structure of the target skin sample through femtosecond laser three - dimensional reconstruction, and analyzing and quantifying the three - dimensional structure using image - processing software to obtain the dermal ridge size parameters of the target skin sample. As shown in Figure 3 The skin structure obtained through femtosecond laser three - dimensional simulation shows a clear dermal ridge structure at the junction of the skin epidermis and dermis.

[0083] S3, preparing a micro - needle mold according to the dermal ridge size parameters, and 3D printing a micro - needle mold with a dermal ridge structure (with a micro - needle height of 200 μm, a width of 150 μm, and a micro - needle gap of 200 μm) using PDMS material.

[0084] S4. Prepare the needle tip solution by adding GelMA and HAMA to the Epilife culture medium containing LAP and fully dissolving them to obtain the needle tip solution. By weight percentage, the needle tip solution includes 0.25% LAP, 10% GelMA, 10% HAMA, and 79.75% Epilife culture medium.

[0085] Prepare the substrate solution by adding GelMA to the Epilife culture medium containing LAP and fully dissolving them to obtain the substrate solution. By weight percentage, the substrate solution includes 0.25% LAP, 10% GelMA, and 89.75% Epilife culture medium.

[0086] Add epidermal stem cells in the logarithmic growth phase to the needle tip solution to obtain a mixed solution. The concentration of epidermal stem cells in the logarithmic growth phase in the mixed solution is 0.5×10 7 / mL

[0087] S5. Fill the mixed solution into the microneedle area of the microneedle mold, perform centrifugation at a temperature of 4°C, set the rotation speed of centrifugation to 1000 rpm, and set the centrifugation time to 5 min. Then fill the substrate solution into the substrate area of the microneedle mold, perform light cross-linking, and demold to obtain the skin ridge-like microneedles loaded with epidermal stem cells. Then place the skin ridge-like microneedles loaded with epidermal stem cells in the Epilife culture medium and culture them for 24 hours at a temperature of 37°C and a carbon dioxide concentration of 5%.

[0088] See Figure 4 As shown, through microscopic observation, it can be seen that epidermal stem cells stably survive at different levels (bottom, middle, and top) of the skin ridge-like microneedle structure.

[0089] Analyze the effects of the prepared cell-loaded skin ridge-like microneedles on wound healing and the reconstruction of the skin ridge structure, specifically including: Randomly divide 48 male nude mice aged 7 - 8 weeks into four groups, namely the control group, comparison group 1, comparison group 2, and experimental group. The mice are raised in a standard SPF room, and the experiments comply with ethical norms and animal welfare requirements. The mice are anesthetized by intraperitoneal injection of sodium pentobarbital solution (1%, W / W), and two full-thickness circular skin wounds with a diameter of 8 mm are created on the back of each mouse.

[0090] The control group is a blank control group, that is, no treatment is given to the skin wounds.

[0091] In comparison group 1, the skin ridge-like microneedles not loaded with epidermal stem cells are transplanted onto the skin wounds.

[0092] In comparison group 2, 3×10 5 epidermal stem cells are transplanted onto each square centimeter of the wound.

[0093] The experimental group transplanted the skin ridge micro - needles loaded with epidermal stem cells prepared in the above example onto the wound per square centimeter.

[0094] All groups were wrapped with sterile gauze and supplemented with sterile normal saline to keep the wound moist. On the 7th, 14th, and 21st days, the wound conditions were recorded and samples were collected for histochemical staining (HE staining). The histochemical staining samples were fixed in 4% (w / v) paraformaldehyde solution, dehydrated, and prepared into paraffin samples, and then sectioned (thickness 6 μm).

[0095] The wound healing rate was statistically analyzed using imageJ software. The formula for calculating the wound healing rate is:

[0096]

[0097] See Figure 5 As shown, by recording the wound conditions, on the 7th and 14th days, the unhealed wound areas of control group 1, control group 2, and the experimental group were all smaller than those of the control group; on the 21st day, the wounds of all four groups were completely healed.

[0098] See Figure 6 As shown, by statistically analyzing the wound healing rate using imageJ software, on the 7th and 14th days, the wound healing rates of control group 1, control group 2, and the experimental group were significantly higher than those of the control group, and there was no significant difference among the wound healing rates of control group 1, control group 2, and the experimental group.

[0099] See Figure 7 As shown, by histochemical staining (HE staining) of the tissue samples on the 21st day, it can be seen that there are no human - like skin ridge specific structures in the control group and control group 1; both control group 2 and the experimental group have human - like skin ridge specific structures, but the skin ridge structure of the experimental group is more obvious than that of control group 2.

[0100] The results show that the skin ridge micro - needles loaded with epidermal stem cells prepared by the preparation method of the present invention have the functions of reconstructing the skin ridge structure of the wound surface and rapidly repairing the wound surface. Since epidermal stem cells are loaded on the bionic micro - needles, the skin ridge micro - needles have the functions of promoting wound epithelialization and restoring the epidermal skin ridge structure, thereby improving the quality of wound healing.

[0101] The above embodiments are only preferred embodiments cited to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention.

Claims

1. A preparation method of a skin ridge micro-needle loaded with epidermal stem cells, characterized in that, Comprising: Providing a target skin sample; Obtaining the three-dimensional structure of the target skin sample through femtosecond laser three-dimensional reconstruction, analyzing and quantifying the three-dimensional structure using image processing software, and obtaining the dermal ridge size parameters of the target skin sample; Preparing a microneedle mold based on the dermal ridge size parameters; Preparing a tip solution and a substrate solution respectively, adding epidermal stem cells in the logarithmic growth phase to the tip solution to obtain a mixed solution; Filling the mixed solution into the microneedle region of the microneedle mold and performing centrifugation; Then filling the substrate solution into the substrate region of the microneedle mold, performing photo-crosslinking, and demolding to obtain dermal ridge-like microneedles loaded with epidermal stem cells.

2. The preparation method of the skin ridge micro-needles loaded with epidermal stem cells according to claim 1, characterized in that, The preparation of the epidermal stem cells in the logarithmic growth phase comprises: Taking the target skin sample, digesting and separating it, removing the dermal tissue, and retaining the epidermal tissue; Crushing, digesting, filtering, and centrifuging the epidermal tissue to collect cells; Adjust the cell concentration to (1 - 2)×10 6 cells / 25 cm 2 , and inoculate them into a culture flask coated with type IV collagen to obtain epidermal stem cells.

3. The preparation method of the artificial dermal ridge microneedles loaded with epidermal stem cells according to claim 1, wherein: The concentration of epidermal stem cells in the logarithmic growth phase cultured in the mixed solution is (0.2 - 0.5) × 10 7 / mL.

4. The preparation method of the skin ridge micro-needles loaded with epidermal stem cells according to claim 1, characterized in that The rotation speed of the centrifugation is set to 800 - 1500 rpm, and the centrifugation time is set to 2 - 7 min.

5. The preparation method of the skin ridge micro-needles loaded with epidermal stem cells according to claim 1, wherein: By weight percentage, the tip solution comprises 0.1 - 0.25% LAP, 5 - 15% GelMA, 5 - 15% HAMA, and 69.75 - 89.9% culture medium; By weight percentage, the substrate solution comprises 0.1 - 0.25% LAP, 5 - 15% GelMA, and 84.75 - 94.9% culture medium.

6. The preparation method of the skin ridge micro-needles loaded with epidermal stem cells according to claim 1, characterized in that, Also comprising: After obtaining the dermal ridge-like microneedles loaded with epidermal stem cells, placing the dermal ridge-like microneedles loaded with epidermal stem cells in a culture medium and culturing them for 24 - 72 hours under the conditions of a temperature of 37°C and a carbon dioxide concentration of 5%.

7. A dermal ridge micro-needle loaded with epidermal stem cells, characterized in that: Prepared by the method for preparing dermal ridge-like microneedles loaded with epidermal stem cells according to any one of claims 1 - 6, comprising a substrate, a tip portion connected to the substrate, and epidermal stem cells loaded on the tip portion.

8. Use of the dermal ridge-like microneedles loaded with epidermal stem cells according to claim 7 in a skin wound repair device.

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