Corneal limbus stem cell in-situ repair microneedle array patch as well as preparation method and application thereof

Abstract

The invention provides a corneal limbus stem cell in-situ repair microneedle array patch and a preparation method and application thereof.The corneal limbus stem cell in-situ repair microneedle array patch comprises a biocompatible backing layer and a microarray layer arranged on the biocompatible backing layer, microneedles of the microarray layer are of a conical structure, the height is 100-300 micrometers, the vertex angle is smaller than 30 degrees, and the thickness of the microneedles of the microarray layer is 10-30 micrometers. And a nano-scale porous structure for loading cornea stem cell suspension is arranged in the cornea stem cell support. Through precise geometric design, biomimetic material selection and spatial arrangement optimization, in-situ delivery, microenvironment activation and tissue fusion in a corneal limbus area are realized under the condition of not depending on in-vitro amplification and surgical suture, and the method becomes an important direction for promoting LSCD minimally invasive treatment strategy innovation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical devices, and particularly relates to a limbal stem cell in-situ repair microneedle array patch, a preparation method thereof and application thereof in treating limbal stem cell deficiency. BACKGROUND

[0002] Limbal stem cell deficiency (LSCD) is an important cause of persistent corneal epithelial defects, impaired ocular surface barrier function, and progressive visual loss. The existing clinical treatment strategies mainly include corneal limbal stem cell ex vivo expansion transplantation (CLET) and small limbal stem cell transplantation (SLET). Among them, CLET relies on a complex in-vitro culture system, and the operation process is complicated, long cycle, and requires a large area of donor tissue, which has high technical threshold and heavy economic burden. SLET, although it avoids the cell expansion process and simplifies the operation, still needs to cut multiple pieces of limbal tissue and adhere and fix them with biological glue or sutures, which has the limitations of low utilization rate of donor tissue, poor adhesion stability, and uneven spatial distribution.

[0003] At the same time, studies have shown that the occurrence of LSCD is not only related to the decrease in the number of stem cells, but also related to the "functional silence" state of the stem cells, and the abnormal local microenvironment may inhibit the regenerative potential of stem cells. Microneedle technology, as a new type of minimally invasive biological delivery means, has the advantages of precise penetration of the epithelium, local controlled release, and controllable structure, and has been widely used in skin beauty, tissue repair and other fields in recent years to activate the function of local stem cells, and has good biological stimulation and regeneration induction potential. Related studies suggest that microneedle puncture can activate quiescent stem cells through slight mechanical stimulation, and then start the tissue regeneration process.

[0004] Based on this, a biodegradable microneedle patch is used as an in-situ delivery platform for limbal stem cells, combined with a small amount of autologous or relative limbal tissue (about 2mm x 2mm) prepared into a cell suspension by enzyme digestion, the stem cells are precisely loaded into the highly customized microneedle structure, and the patch is attached to the limbal defect area, which can avoid the dependence on large volume donor tissue in traditional operation, and can realize stable anchoring and in-situ activation of cells without suturing or biological glue, which has significant minimally invasive and clinical promotion value.

[0005] Based on the application, the prior art also makes similar explorations, such as US20220062606A1, which discloses a microneedle loaded with living cells, prepared by low-temperature freeze-drying technology, and the patch "melts" to release after injection, but its treatment purpose is completely different from the present application and is completely unrelated to the treatment of the corneal limbus; US8636713B2 discloses a hollow microneedle for the eye, which delivers high-concentration drugs through injection, but it is only suitable for the posterior segment of the corneal limbal area and is not suitable for the anterior segment of the corneal limbal area. In addition, according to the existing literature, the current microneedle patch for the eye is limited to drug or growth factor delivery, and there is a lack of attention to the in-situ activation and colonization of stem cells.

[0006] Current studies have confirmed that microneedle systems constructed from natural polysaccharides such as hyaluronic acid (HA) and chitosan (CS) exhibit excellent biocompatibility, controllable hydration properties, and degradation performance in corneal tissue. HA microneedles can simulate the moist environment of the corneal basement membrane to promote cell survival, and CS can enhance adhesion to the ocular mucosa through its cationic structure and has natural antibacterial activity, reducing the risk of postoperative infection. However, most current microneedle systems still face several technical challenges when applied to LSCD: first, the needle height, sharpness, and corneal tissue penetration are not well matched; second, it is difficult to balance the structural strength and degradation rate, which may result in structural collapse or shedding during delivery; third, the spatial distribution of stem cells in the microneedle is uneven, limiting the efficiency of colonization and functional integration. SUMMARY

[0007] To address the above problems, the present application designs a microneedle array patch for in-situ repair of limbal stem cells (LSCs). Previous studies have shown that conical microneedles with a height of 100-300 μm and an apex angle of less than 30° can effectively improve the penetration efficiency of the corneal epithelium and avoid damage to the stroma. Combined with advanced microfabrication techniques such as two-photon polymerization and 3D printing, it is expected to achieve a microneedle array system with consistent structure and scalable production. At the same time, the internal nano-porous structure of the microneedle helps to improve the loading capacity of stem cells. Therefore, the present application develops a polysaccharide microneedle patch that can load limbal stem cell suspension. Through precise geometric design, selection of biomimetic materials, and optimization of spatial arrangement, the patch can achieve in-situ delivery, microenvironment activation, and tissue integration in the corneal limbal area without relying on in-vitro expansion and surgical suturing, becoming an important direction for promoting the innovation of minimally invasive treatment strategies for LSCD.

[0008] To achieve the above purpose, the specific technical solutions adopted by the present application are as follows:

[0009] In the first aspect of the present application, a limbal stem cell in situ repair microneedle array patch is provided, which comprises a biocompatible backing layer and a microneedle array layer arranged thereon, and further comprises an optional growth factor sustained-release module. The specific structure of each part is described as follows:

[0010] (1) Biocompatible backing layer

[0011] The biocompatible backing layer is prepared from medical-grade polyvinyl alcohol (PVA) or methyl cellulose material, with a thickness of 50-100 μm, and a 1-2 mm positioning grid is printed on the surface to assist ophthalmic positioning operation. The edge is provided with a tear-triggered degradable adhesive ring composed of low molecular weight HA (<10 kDa), which can provide an adhesive force of 1-5 mN to ensure the stability of the patch on the corneal surface after fitting.

[0012] (2) Growth factor sustained-release module

[0013] It is selected to be embedded in PLGA and / or gelatin electrospun nanofiber membrane (diameter 200-500 nm), and loaded with PLGA encapsulated bFGF sustained-release microspheres with a particle size of 1-3 μm, to realize enzyme-responsive and sustained growth factor delivery for 3-7 days.

[0014] (3) Microneedle array layer

[0015] The whole is a thin layer, curved spherical rectangular arc-shaped strip, the inner and outer edges are parallel to the limbus, and the two ends are straight edges (or slightly rounded) perpendicular to the tangent of the limbus, which is suitable for fitting the local limbal lesion area. For the damaged limbal part of the patient, the patch part can be the temporal superior quadrant, the nasal inferior quadrant, etc., and the angle range can be any size of angle or clock position (recommended as 45°-90°), which needs to be strictly designed and adjusted according to the preoperative data of the patient. The arc length L of the microneedle array layer can be adjusted according to the angle range, the horizontal diameter and vertical diameter of the cornea, the width W is designed according to the size of the damaged cornea of the patient, and the thickness T is recommended to be 0.1-0.2 mm which is thin and has certain strength. The specific shaping of the patch can also adjust the microneedle arrangement and needle height.

[0016] Specifically, the microneedle array layer comprises a bearing substrate and a plurality of microneedles arranged thereon at a certain density, each microneedle is provided with a nanoscale porous structure for loading limbal stem cell suspension and sustained-release growth factor combination.

[0017] Preferably, the arrangement density of the microneedle array is 40-50 roots / mm 2, the microneedle is in a conical structure, the height is 100-300 μm, and the top angle is less than 30°. Further preferably, the length of a single microneedle is controlled to be 200 μm ± 20 μm, the diameter of the bottom of the needle is 100 μm, the base needle pitch is set to be 150 μm, the needle is perpendicular to the base, the needle tip angle is 25° ± 3°, and the corneal epithelial layer can be effectively penetrated without damaging the tissues below the Bowman membrane.

[0018] In terms of the microneedle structure, a nanoscale porous structure is arranged in the middle of each microneedle, the pore size is concentrated in 50-100 nm (preferably 70 ± 10 nm), the pore volume is 0.5-0.8 cm 3 / g, and is used for loading a corneal limbal stem cell suspension (cell density 1×10 6 cells / mL) and a slow-release growth factor combination (such as 0.1 μg / μL of bFGF and 0.05 μg / μL of EGF). The preparation method of the corneal limbal stem cell suspension is as follows: autologous or relative donor corneal limbal tissue with a small amount of material (about 2 mm×2 mm) is used, and a cell suspension is prepared through enzyme digestion.

[0019] The nanoscale porous structure can be formed in various ways, such as a 3D printing way, a microneedle array structure containing a pore size of 50-100 nm is constructed by controlling the layer thickness and the light curing time of each layer, or the microneedle array structure can be constructed through freeze-drying technology or emulsion evaporation method.

[0020] In terms of the preparation material, the microneedle array layer is prepared from a hyaluronic acid (HA) and chitosan (CS) composite polysaccharide material, and the mass ratio between the two is 1:1-3:1. The HA provides high hydrophilicity and ocular surface moisturizing function, maintains the local hydration state, the CS has cationic properties and antibacterial ability, and enhances the adhesion.

[0021] In the second aspect of the present application, a preparation method of the above-mentioned corneal limbal stem cell in-situ repair microneedle array patch is provided, and the preparation method is outlined as follows: the hyaluronic acid (HA) and chitosan (CS) are compounded according to the mass ratio, the mechanical properties and the degradation rate are regulated by a crosslinking agent (such as glutaraldehyde or an ionic crosslinking agent), the autologous or relative donor corneal limbal tissue with a small amount of material (about 2 mm×2 mm) is used, a cell suspension is prepared through enzyme digestion, and then the cell suspension is directly loaded into the highly customized biodegradable microneedle array patch, the HA provides high hydrophilicity and ocular surface moisturizing function, the CS has cationic properties and antibacterial ability, and enhances the adhesion. The microneedle is manufactured through a hot die pressing or micromolding process, the preset mold is used to control the geometric morphology of the microneedle, and a highly consistent three-dimensional conical structure array is formed. The specific preparation method is as follows:

[0022] A, microneedle array layer preparation

[0023] After mixing the hyaluronic acid and chitosan complex polysaccharide material to form a homogeneous gel, the crosslinking agent and the corneal limbal stem cell suspension and growth factor mixed solution are mixed, and the SLA light curing 3D printing or PDMS mold injection molding technology is used to precisely adjust the mechanical strength and porosity, to form a porous microneedle array with an elastic modulus of 10-50 kPa and a degradation time of 1-2 hours, which meets the mechanical and degradation requirements of corneal tissue.

[0024] Preferably, the crosslinking agent is selected from 0.5%-1% methacrylate crosslinking agent or 1%-5% glutaraldehyde.

[0025] When SLA light curing 3D printing is used for preparation, the preparation method is as follows: hyaluronic acid and chitosan are added to sterile PBS buffer according to a mass ratio of 1:1-3:1, and slowly stirred to form a homogeneous gel, while 0.5%-1% methacrylate crosslinking agent is added for subsequent light curing shaping; then, 100-200 μL of corneal limbal stem cell and growth factor mixed suspension is mixed with the gel, and the mixture is injected into the PDMS mold through the 3D printer, the printing parameters are adjusted, the layer thickness is limited to 15-25 μm, the light curing time is 30-60 seconds per layer, and a microneedle array structure containing 50-100 nm pore size is constructed.

[0026] When the PDMS mold injection molding technology is used, the preparation method is as follows: the hyaluronic acid and chitosan homogeneous gel is mixed with 1%-5% glutaraldehyde, and injected into the PDMS mold using a conventional method to form a hollow microneedle array, then a predetermined amount of corneal limbal stem cell suspension and growth factor mixed solution is injected into the microneedle inner hole by vacuum negative pressure injection method (vacuum-assisted loading), and a microneedle array structure containing 50-100 nm pore size is constructed by 4°C freeze drying technology or emulsion evaporation technology to form a stable dry powder patch, which can be activated to form a gel-like release matrix by using physiological saline.

[0027] In addition to freeze-drying to prepare nanoscale porous structures to load corneal stem cell suspension, other forms of microneedle loading corneal stem cells can also be used. For example, the corneal stem cell suspension is mixed with the above hydrogel material directly, and cast into a solid microneedle array patch. The capacity of the corneal stem cell suspension is 1-3 μL for solid microneedles, and about 0.15-0.5 μL for porous microneedles.

[0028] B, in situ repair of microneedle array patch preparation

[0029] A piece of medical grade PVA backing film with a thickness of 50-100 μm is cut, a low molecular weight HA (<10 kDa, concentration of 2%-5%) adhesion layer is coated on its surface by spraying method, then the formed microneedle array is carefully peeled off from the mold and attached to the backing film vertically, ensuring that the microneedle arrangement direction is outward, and a positioning gap is reserved at the edge after attachment, which is used to align the limbal defect boundary in clinical application; after assembly, low temperature plasma with a power of 50 W and a time of 15 minutes is used for sterilization, and the environmental humidity is kept <30% during the process to prevent material moisture deformation, and after sterilization, the patch is stored in a 4-8℃ refrigerated environment for standby.

[0030] In order to ensure the accurate attachment between the microneedle array and the PVA backing film, a positioning center hole can be designed on both of them.

[0031] In the third aspect of the present application, a limbal stem cell deficiency treatment assembly is provided, which comprises the above-mentioned limbal stem cell in-situ repair microneedle array patch and a contact lens carrier used in cooperation therewith. The curvature radius of the contact lens carrier is 7.8-8.5 mm, which is used to protect the microneedle patch or moisturize the treatment after the backing layer of the patch is removed.

[0032] During operation, the backing layer of the patch can be clamped, and the limbal defect area is accurately aligned with the help of a surgical microscope. The microneedle insertion process is completed within about 10 seconds after the ocular surface is attached to the microneedle patch, and the microneedle body is gradually absorbed in 15-30 minutes to start dissolving. The stem cells are released to the limbal basement layer through the rehydration of the porous structure, and are maintained in a survival state in the gel scaffold formed by polysaccharide degradation, realizing local adhesion and biological integration, and realizing efficient planting by means of electrostatic adsorption between CS cations and cell surface glycoproteins. The cationic surface of chitosan forms electrostatic adsorption with the anionic mucus layer of the ocular surface after the microneedle is inserted, which helps the stem cells to adhere in-situ in the limbal basement layer, and improves the initial planting efficiency.

[0033] The release of bFGF and EGF reaches a peak within the first 24 hours, and then continues to release for 72 hours, simulating the physiological gradient of cytokines in the natural injury repair process. The backing layer after attachment can be naturally removed, or removed by physiological saline cleaning, which is simple and convenient to operate without surgery, and is suitable for primary medical institutions.

[0034] The experimental results show that the HA matrix maintains a local hydration state, the CS provides antibacterial protection (bacteriostatic rate > 90%), and the degradation product glucosamine also helps to synthesize the extracellular matrix. The overall technical indicators include precisely controlling the microneedle penetration depth to be 50-100 mu m, ensuring that the corneal stroma layer is not damaged; the in-vitro survival rate of stem cells is > 95%, and the in-vivo planting efficiency is improved by 30%-50% compared with traditional surgery; the growth factor release curve shows that the total amount released in the early stage of 0-24 hours accounts for 30%-40%, and the daily release is maintained at 5%-10% in the later stage, providing stable support for tissue repair. The overall scheme integrates biomaterial engineering and ophthalmic clinical needs, and has precision, safety and practicality.

[0035] In summary, the microneedle array patch of the present application can meet the individualized repair needs of different ocular surface damage areas, and achieves:

[0036] (1) The microneedle array is regularly arranged to simulate the nest-like structure of the corneal limbal stem cells;

[0037] (2) The HA / CS polysaccharide matrix is homologous to the corneal tissue, and has good biocompatibility and antibacterial properties;

[0038] (3) The patch can be attached to the ocular surface by a contact lens carrier to achieve non-invasive delivery;

[0039] (4) The microneedle dissolves to release degradation products, forming a temporary scaffold to guide epithelial cell regeneration to cover the damaged area.

[0040] Compared with the prior art, the beneficial technical effects of the present application are as follows:

[0041] The microneedle array patch exhibits significant efficiency and safety improvement in the in-situ repair of corneal limbal stem cells, and has systematic advantages in surgical process optimization, tissue repair quality, patient benefits and medical resource utilization compared with traditional corneal limbal stem cell transplantation such as CLET, SLET and other ocular surface repair methods. Its innovation lies in the integration of prefabricated microneedle array and corneal contact lens carrier, and the preloading of stem cells and growth factors in the controllably degradable porous microneedle structure, which converts the complex steps of separation, culture and transplantation into a single step of attachment operation, reducing the operation time by more than 60% compared with CLET, and is suitable for popularization in primary ophthalmology.

[0042] In terms of structure, regular arrangement (40-50 roots / mm2) and standardized conical geometry (needle length 100-300 mu m) can ensure that the stem cells are accurately delivered to the basal layer of the corneal limbus, with a positioning error controlled within 100 mu m, which is significantly better than traditional transplantation (> 500 mu m), improving the consistency and controllability of tissue repair. Compared with traditional transplantation, the use of microneedle patch to deliver corneal stem cells can stimulate the patient's own silent corneal stem cells, promote self-repair and improve efficacy.

[0043] The patch body material is a hyaluronic acid / chitosan composite polysaccharide, which is highly homologous to the components of corneal tissue, has good biocompatibility and postoperative fusion capacity, reduces the incidence of inflammation by 40% compared with traditional surgery, and chitosan has natural antibacterial properties, which reduces the risk of postoperative infection by more than 50%.

[0044] The 50-100 nm pore channel constructed inside the microneedle can form a biomimetic “stem cell nest” microenvironment, and the in-vitro survival rate of stem cells can be maintained for more than 95% within 24 hours, the in-vivo colonization rate is increased by 30%-50% compared with SLET, and the repair of corneal epithelial defects is accelerated by about 20%-30%. In terms of patient benefits, the microneedle can be completely degraded within 1-2 hours, without the need for postoperative removal, avoiding implant-related damage, and the patient's pain score (VAS) is reduced by 2-3 points (full score of 10 points) compared with CLET, and the entire treatment program does not rely on in-vitro expansion systems or immunosuppressants, and the material cost is only 10%-20% of CLET, significantly reducing the economic burden. Its manufacturing process uses green micro-fermentation raw materials and non-toxic cross-linking technology, and the degradation products of the product are monosaccharides and glucosamine that can be naturally metabolized, which is environmentally friendly and reduces medical waste by more than 70%.

[0045] At the same time, by adjusting the cross-linking degree and drug loading type of the microneedle, it can adapt to different types of LSCD, including traditional surgery contraindicated groups such as chemical burns, dry eye or systemic anticoagulant therapy patients, greatly expanding the applicable population. In summary, the microneedle array patch takes “precise delivery-biomimetic colonization-safe degradation” as the core technical path, and has the advantages of efficient repair, minimally invasive operation and resource sustainability, providing a scalable clinical solution for limbal stem cell regeneration medicine, and has broad application prospects and transformation potential. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 A structure schematic diagram of the limbal stem cell in-situ repair microneedle array patch of the application is shown;

[0047] Figure 2 A local structure enlarged schematic diagram of the limbal stem cell in-situ repair microneedle array patch of the application is shown;

[0048] Figure 3 A scanning electron microscope diagram of the limbal stem cell in-situ repair microneedle array patch prepared by the application is shown;

[0049] Figure 4 A microneedle cross-sectional structure schematic diagram is shown;

[0050] Figure 5 A microneedle cross-sectional structure schematic diagram loaded with stem cells is shown;

[0051] Figure 6The use state schematic diagram of the limbal stem cell in-situ repair microneedle array patch of the application is shown, and the example patch is located in the temporal superior quadrant of the right cornea.

[0052] Wherein, ① backing layer, ② microneedle, ③ microneedle needle hole. DETAILED DESCRIPTION

[0053] The following examples and experimental examples further illustrate the application and are not to be construed as limiting the application.

[0054] The innovations of the application are as follows:

[0055] ① The application significantly improves the spatial resolution and directional control of stem cell delivery by precisely designing the geometric parameters of the microneedle array (length 100-300 μm, needle tip angle 20°-30°), solves the problems of low preparation precision and inaccurate positioning of traditional manual grafts, and realizes minimally invasive and directional delivery in the limbal region.

[0056] ② Using the microneedle patch to deliver corneal stem cells not only injects new corneal stem cells into the patient's corneal defect site, but also stimulates the patient's own silent corneal stem cells, which is more advantageous than traditional manual grafts, and promotes the patient's corneal repair from two aspects.

[0057] ③ The biodegradable polysaccharide matrix constructed by hyaluronic acid (HA) and chitosan (CS) has excellent biocompatibility (homologous to the corneal extracellular matrix) and mechanical adaptability (elastic modulus 10-50 kPa), which can effectively simulate the natural limbal microenvironment and reduce the immune response.

[0058] ④ The microneedle is internally constructed with a porous network of 50-100 nm pore size for loading stem cells and growth factors such as bFGF and EGF, achieving early rapid release to promote cell adhesion and late sustained release to maintain proliferation signals, overcoming the burst release effect and insufficient release problem of traditional delivery systems.

[0059] ⑤ The microneedle array has a density of about 44 roots / mm², which simulates the spatial distribution pattern of the limbal stem cell nest, and the electrostatic adsorption ability of the chitosan cationic surface to the ocular mucosa, improving the homing efficiency and stability of in-situ planting of stem cells, breaking through the technical bottleneck of random distribution of traditional transplantation.

[0060] ⑥ The microneedle is prepared by 3D printing or micromolding process, with controllable crosslinking density, and the dissolution time is completed within 1-2 hours, avoiding the risk of implantation residue, and the degradation products form a temporary scaffold to support epithelial cell migration and tissue regeneration.

[0061] ⑦ The patch achieves non-invasive adhesion by relying on a contact lens carrier structure, without the need for microsurgery. It is suitable for primary healthcare settings, significantly reducing the surgical threshold and overall treatment cost, and has good potential for promotion and commercialization.

[0062] The microneedle array patch for in situ repair of limbal stem cells in this invention consists of three parts: a polysaccharide microneedle array layer, a biocompatible backing layer, and an optional growth factor sustained-release module. The overall design takes into account precise delivery, biomimicry, and clinical operability.

[0063] The microneedle array utilizes a composite polysaccharide material of hyaluronic acid (HA) and chitosan (CS). After modification by methacrylate esterification, a cross-linking reaction is performed to form a three-dimensional scaffold with an elastic modulus of 10–50 kPa and a degradation time of 1–2 hours, meeting the mechanical and degradation requirements of corneal tissue. Each microneedle has a conical structure with a height controlled at 100–300 μm (close to the thickness of the corneal epithelium) and an apex angle of less than 30° to ensure penetration of the corneal epithelial barrier without reaching the stromal layer. The microneedles internally incorporate a porous structure with a pore size of 50–100 nm to simulate the spatial distribution of limbal stem cell nests, used to load limbal stem cell suspensions (concentration 1×10⁻⁶). 6 ~5×10 6 Microneedles are prepared using SLA photopolymerization 3D printing or PDMS mold injection molding technology, with 1% to 5% glutaraldehyde to control the crosslinking density, thereby precisely adjusting mechanical strength and porosity. The microneedles contain growth factors (such as bFGF 10~50 ng / mL and EGF 5~20 ng / mL) and cells / mL, enabling simultaneous release immediately after implantation.

[0064] In a specific implementation of this invention, a microneedle array is prepared using 3D printing, as follows:

[0065] Before production, prepare polysaccharide raw materials (hyaluronic acid HA and chitosan CS), limbal stem cell suspension, PDMS microneedle mold, 3D bioprinter and medical-grade PVA backing film.

[0066] First, HA and CS are added to sterile PBS buffer at a mass ratio of 1:1–3:1 and slowly stirred until a homogeneous gel is formed. At the same time, 0.5%–1% of methacrylate crosslinking agent is added for subsequent photocuring and shaping. Next, a quantitative amount of limbal stem cell suspension (100–200 μL) is mixed with the gel and injected into a PDMS mold using a 3D printer. The printing parameters are adjusted (e.g., layer thickness 20 μm, photocuring time 30–60 seconds / layer) to construct a microneedle array structure with pore size of 50–100 nm.

[0067] Then cut a piece of medical grade PVA backing film with a thickness of 50-100 μm, and use a spray method to coat a low molecular weight HA adhesion layer (molecular weight <10 kDa, concentration 2%-5%) on its surface, then carefully peel the formed microneedle array from the mold, and vertically adhere it to the backing film, ensuring that the microneedle arrangement direction is outward, and after the adhesion is completed, a positioning gap with a diameter of 1-2 mm is reserved at the edge, which is used to align the limbal defect boundary in clinical application.

[0068] After the patch assembly is completed, it is placed in a sterile culture dish, and sterilized using low temperature plasma with a power of 50 W and a time length of 15 minutes, and attention is paid to keep the environmental humidity <30% during the process to prevent material moisture deformation; after sterilization is completed, the patch is stored in a 4-8°C refrigeration environment for standby, and thus a limbal stem cell microneedle array patch with uniform specifications and accurate structure is prepared.

[0069] Figure 3 An enlarged physical diagram of the limbal stem cell in-situ repair microneedle array patch prepared in the embodiment is shown; Figure 5 An illustration of the microneedle array patch located in the temporal superior quadrant prepared by the above method is shown. In actual use, the angle range thereof is 0:30-2:30 (60°), the arc length L=6 mm, the width (the radial width of the patch extending from the limbus to the corneal center) W=2 mm, and the thickness T=0.1 mm. The patch has 39 rows of microneedles in the arc length direction, and 13 columns of microneedles in the width direction, and a total of 507 microneedles. The patch capacity is about 1.20 μL if solidly absorbed, and about 0.19 μL if hollow.

[0070] When using the microneedle patch, attention should be paid to only holding the backing layer to avoid holding the microneedle patch and damaging the microneedle structure, and the backing layer can be naturally removed after the microneedle is partially dissolved or completely dissolved in the corneal stroma layer, and a scleral lens with a suitable size is worn according to the patient's condition for protection.

[0071] The unexplained parts involved in the present application are the same as or realized by the prior art. The applicant declares that the present application is illustrated by the above embodiments to illustrate the detailed method of the present application, but the present application is not limited to the above detailed method, that is, it does not mean that the present application must rely on the above detailed method to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. A limbal stem cell in situ repair microneedle array patch, characterized by, The microarray layer is loaded with corneal stem cells on the biocompatible backing layer, The microneedle is in a conical structure, with a height of 100-300 μm and a top angle less than 30°.

2. The limbal stem cell in-situ repair microneedle array patch according to claim 1, wherein: the biocompatible backing layer and the microarray layer are provided with a growth factor sustained-release layer, which is an embedded PLGA and / or gelatin electrospun nanofiber membrane loaded with PLGA encapsulated bFGF sustained-release microspheres. wherein The diameter of the electrospun nanofiber membrane is 200-500 nm, and the particle size of the sustained-release microspheres is 1-3 μm.

3. The limbal stem cell in-situ repair microneedle array patch according to claim 1, wherein: the biocompatible backing layer is prepared from medical-grade polyvinyl alcohol or methylcellulose material, with a thickness of 50-100 μm and a surface printed with a 1-2 mm positioning grid, and the edge is provided with a tear-triggered degradation adhesive ring composed of low molecular weight HA.

4. The limbal stem cell in-situ repair microneedle array patch according to claim 1, wherein: the microarray layer is prepared from hyaluronic acid and chitosan composite polysaccharide material, and includes a bearing substrate and a plurality of microneedles arranged thereon at a certain density, each microneedle being provided with a nanoscale porous structure for loading a limbal stem cell suspension and a sustained-release growth factor combination. wherein 5. The limbal stem cell in-situ repair microneedle array patch according to claim 4, wherein: the length of a single microneedle is controlled to be 200 μm ± 20 μm, the diameter of the needle bottom is 100 μm, the base needle pitch is set to be 150 μm, the needle is perpendicular to the base, and the needle tip angle is 25° ± 3°.

6. The limbal stem cell in-situ repair microneedle array patch according to claim 4, comprising the following steps: wherein, A. Preparation of the microarray layer After mixing the hyaluronic acid and chitosan composite polysaccharide material to form a homogeneous gel, the crosslinking agent and the limbal stem cell suspension and growth factor mixture are mixed, and SLA photocuring 3D printing or PDMS mold injection molding technology is used to form a porous microneedle array with an elastic modulus of 10-50 kPa and a degradation time of 1-2 hours; wherein The shape of the microarray layer is a spherical rectangular arc-shaped patch, the angle range is 45°-90°, and the arrangement density of the microneedle array is 40-50 roots / mm 2 , B. Preparation of the in-situ repair microneedle array patch A piece of medical-grade PVA backing film is cut, a low molecular weight HA adhesive layer is coated on the surface thereof by spraying, then the formed microneedle array is carefully peeled off from the mold, and is vertically attached to the backing film to ensure that the microneedle arrangement direction is outward, and a positioning notch is reserved at the edge after the attachment is completed, which is used to align the limbal defect boundary during clinical application; after the assembly is completed, sterilization is performed in a certain humidity environment, and after sterilization is completed, the patch is stored in a 4-8℃ refrigerated environment for standby. wherein The nanoscale porous structure is arranged in the middle and lower part of each microneedle, the pore size is concentrated in 50-100 nm, and the pore volume is 0.5-0.8 cm 3 / g; in the loaded conjunctival stem cell suspension, the stem cell density is 1-5x10 6 cells / mL, and the sustained-release growth factor combination consists of 0.1 μg / μL of bFGF and 0.05 μg / μL of EGF.

7. The method of claim 1-6, wherein the limbal stem cell in situ regeneration microneedle array patch is prepared by the steps of:

8. The preparation method of the limbal stem cell in-situ repair microneedle array patch according to claim 7, wherein: In step A, the crosslinking agent is selected from 0.5%-1% methacrylate crosslinking agent or 1%-5% glutaraldehyde. ​ ​ ​ ​ wherein ​ The method for constructing the porous microneedle structure by SLA light curing 3D printing is as follows: hyaluronic acid and chitosan are added to sterile PBS buffer at a mass ratio of 1:1-3:1, and slowly stirred to form a homogeneous gel, while 0.5%-1% of a methacrylate crosslinking agent is added for subsequent light curing shaping; then, 100-200 μL of corneal limbal stem cell and growth factor mixed suspension is mixed with the gel, and the mixture is injected into the PDMS mold by the 3D printer, the printing parameters are adjusted, the layer thickness is limited to 15-25 μm, the light curing time is 30-60 seconds per layer, and the microneedle array structure containing 50-100 nm pore size is constructed; The method for constructing the porous microneedle structure by PDMS mold injection molding technology is as follows: the homogeneous gel of hyaluronic acid and chitosan is combined with 1%-5% glutaraldehyde, and injected into the PDMS mold by a conventional method to form a hollow microneedle array, then a predetermined amount of corneal limbal stem cell suspension and growth factor mixture is injected into the microneedle inner hole by vacuum negative pressure injection method, and a microneedle array structure containing 50-100 nm pore size is constructed by 4°C freeze-drying technology or emulsion evaporation technology.

9. The preparation method of the corneal limbal stem cell in-situ repair microneedle array patch according to claim 7, characterized in that: In step B, the thickness of the medical-grade PVA backing film is 50-100 μm; the HA molecular weight in the low molecular weight HA adhesion layer is <10 kDa, and the concentration is 2%-5%; the diameter of the positioning notch is 1-2 mm, The sterilization conditions are as follows: low-temperature plasma with a power of 50 W and a time length of 15 minutes is used for sterilization, and the environmental humidity is kept <30% during the process to prevent material moisture absorption deformation.

10. A limbal stem cell deficiency treatment assembly comprising, The corneal contact lens carrier is used in cooperation with the corneal limbal stem cell in-situ repair microneedle array patch. The corneal limbal stem cell in-situ repair microneedle array patch is as shown in any one of claims 1-6.

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