Acellular dermal matrix patch, preparation method therefor, and application thereof

Abstract

An acellular dermal matrix patch, a preparation method for the acellular dermal matrix patch, and an application of the acellular dermal matrix patch in repairing tissue damage. The acellular dermal matrix patch comprises a plurality of patch regions, and the patch regions have at least two crosslinking characteristics. The present invention further provides a preparation method for the acellular dermal matrix patch, and an application of the acellular dermal matrix patch in repairing tissue damage.

Description

Dermal acellular matrix patch and its preparation method and application

[0001] Related applications

[0002] This application claims priority to Chinese patent application No. 202410360606.X, filed on March 27, 2024, entitled “Dermal Acellular Matrix Patch, Preparation Method and Application Thereof,” the entire text of which is hereby incorporated by reference. Technical Field

[0003] The present application relates to the field of medical materials, and in particular, to a dermal acellular matrix patch, a method for preparing the dermal acellular matrix patch, and an application of the dermal acellular matrix patch in repairing tissue damage. Background Art

[0004] Many biomaterials, such as collagen, can provide physical scaffold support for cell proliferation, growth, and differentiation after being implanted into the body, thereby promoting the repair of damaged tissues.

[0005] Many of these biomaterials are often biodegradable in the body, but there's often a mismatch between material degradation and tissue repair. For example, scaffold-induced tissue repair typically progresses from the site of direct tissue contact toward the distal end. However, due to rapid material degradation, the scaffold may be completely degraded before tissue repair has progressed to the distal end, leading to repair failure. Alternatively, if the material degrades too slowly, it may remain in the body for too long, potentially causing adverse reactions such as chronic inflammation. Furthermore, the healing process often varies between individuals, for different damaged areas, and for varying degrees of injury.

[0006] Therefore, how to invent a repair material product with degradation properties that can be widely applied to various injuries of various groups of people has always been a major fundamental issue in academia and industry. Summary of the Invention

[0007] The present application aims to solve one of the technical problems in the related art to a certain extent. To this end, the present application provides a dermal acellular matrix patch, a method for preparing the dermal acellular matrix patch, and the use of the dermal acellular matrix patch in repairing tissue damage.

[0008] To achieve the above objectives, as a first aspect of the present application, a dermal acellular matrix patch is provided, wherein the dermal acellular matrix patch comprises a plurality of patch regions, wherein the plurality of patch regions have at least two cross-linking properties.

[0009] In some embodiments, at least one of the plurality of said patch regions is an uncrosslinked patch region.

[0010] In some embodiments, the plurality of patch regions include a central region and a peripheral region disposed around the central region.

[0011] In some embodiments, the central area includes at least one first central area and at least one second central area, and the first central area and the second central area are both strip-shaped areas; the length direction of the first central area is consistent with the length direction of the second central area, the cross-linking characteristics of the second central area are the same as the cross-linking characteristics of the peripheral area, and the cross-linking characteristics of the first central area and the cross-linking characteristics of the second central area are different from each other; or the central area includes multiple first central areas and multiple second central areas, and the length direction of the first central area is inconsistent with the length direction of the second central area, so that the first central area and the second central area intersect with each other and are defined as cells; and the cross-linking characteristics of each area of ​​the dermal acellular matrix patch meet the first or second conditions, wherein: the first condition is that the cross-linking characteristics of the first central area are the same as the cross-linking characteristics of the second central area, and are different from the cross-linking characteristics of the cell, and the cross-linking characteristics of the cell are the same as the cross-linking characteristics of the peripheral area; the second condition is that the cross-linking characteristics of the first central area, the cross-linking characteristics of the second central area, and the cross-linking characteristics of the cell are different from each other, and the cross-linking characteristics of the cell are the same as the cross-linking characteristics of the peripheral area.

[0012] In some embodiments, an edge of at least one of the first central area and the second central area is a curved line.

[0013] In some embodiments, the multiple patch areas include at least one first patch area and at least one second patch area, and the first patch areas and the second patch areas are arranged alternately in parallel; or, the multiple patch areas include multiple first patch areas, multiple second patch areas and multiple cell areas, and multiple first patch areas and multiple second patch areas intersect with each other.

[0014] In some embodiments, an edge of at least one of the first patch area and the second patch area is a curved line.

[0015] As a second aspect of the present application, a method for preparing a dermal acellular matrix patch is provided, the preparation method comprising: providing an initial dermal acellular matrix patch; performing patterned cross-linking on the initial dermal acellular matrix patch to obtain the dermal acellular matrix patch, wherein the dermal acellular matrix patch comprises multiple patch areas, wherein the multiple patch areas have at least two cross-linking properties.

[0016] In some embodiments, the patterned cross-linking of the initial dermal acellular matrix patch includes: setting a patterning tool on the surface of the initial dermal acellular matrix patch, the patterning tool including at least one shielding area and at least one hollow area; using a first cross-linking agent to cross-link the initial dermal acellular matrix patch provided with the patterning tool.

[0017] In some embodiments, the blocking area and the hollow area are both in the shape of bars, the patterning tool includes multiple blocking areas and multiple hollow areas, the length direction of the blocking area is consistent with the length direction of the hollow area, and the blocking area and the hollow area are alternately arranged.

[0018] In some embodiments, patterning and cross-linking the initial dermal acellular matrix patch further comprises: removing the patterning tool to obtain an intermediate patch; and cross-linking the intermediate patch using a second cross-linking agent.

[0019] In some embodiments, the initial dermal acellular matrix patch is patterned and cross-linked to obtain the dermal acellular matrix patch, including: setting a first patterning tool on the surface of the initial dermal acellular matrix patch, the first patterning tool including at least one first blocking area and at least one first hollow area, the length direction of the first blocking area is consistent with the length direction of the first hollow area, and the first blocking area and the first hollow area are alternately arranged; using a first cross-linking agent to cross-link the initial dermal acellular matrix patch provided with the first patterning tool; removing the first patterning tool to obtain a first intermediate patch; setting a second patterning tool on the surface of the first intermediate patch, the second patterning tool including at least one second blocking area and at least one second hollow area, the length direction of the second blocking area is consistent with the length direction of the second hollow area, and the second blocking area and the second hollow area are alternately arranged, and the length direction of the second blocking area intersects with the length direction of the first blocking area; using a second cross-linking agent to cross-link the first intermediate patch provided with the second patterning tool.

[0020] In some embodiments, patterning and cross-linking the initial dermal acellular matrix patch to obtain the dermal acellular matrix patch further includes: removing the second patterning tool to obtain a second intermediate patch; and cross-linking the second intermediate patch using a third cross-linking agent.

[0021] As a third aspect of the present application, a use of a dermal acellular matrix patch in repairing tissue damage is provided. The dermal acellular matrix patch is the dermal acellular matrix patch described in the first aspect of the present application.

[0022] The present application provides a dermal acellular matrix patch having at least two different cross-linking properties. The degradation time corresponding to the patch areas with different cross-linking properties is also different. The collagen activity of the patch areas with short degradation time is relatively high, and the collagen activity of the patch areas with long degradation time is relatively low. Therefore, when the dermal acellular matrix described in the present application is implanted into the body to repair damaged tissue, the patch areas with relatively high collagen activity in the dermal acellular matrix patch can repair the damaged area relatively quickly, while the patch areas with relatively low collagen activity have a relatively long retention time in the body and can comprehensively repair the damaged tissue. By providing a patch area including at least two cross-linking properties, the patch areas with different collagen activities can be organically combined, so that the dermal acellular matrix patch has a continuous and stable repair effect.

[0023] These features of the present application will be disclosed in detail in the following detailed description and accompanying drawings. The best embodiments or means of the present application will be fully illustrated in conjunction with the accompanying drawings, but are not intended to limit the technical solutions of the present application. Furthermore, although multiple features, elements, and components may be present in each of the following text and accompanying drawings, they may be labeled with different symbols or numbers for convenience, but all represent components with the same or similar structure or function. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the conventional technology, the following briefly introduces the drawings required for use in the embodiments or the conventional technology descriptions. Obviously, the drawings described below are merely embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the disclosed drawings without any creative work.

[0025] FIG1 to FIG23 are schematic diagrams of 23 embodiments of the dermal acellular matrix patch provided in this application.

[0026] FIG24 is a flow chart of an optional embodiment of the method for preparing a dermal acellular matrix patch provided in an example of the present application.

[0027] FIG25 is a schematic diagram of an optional implementation of step S120.

[0028] 26 is a schematic diagram of one embodiment of a patterning tool.

[0029] 27 is a schematic diagram of another embodiment of a patterning tool.

[0030] FIG28 is a schematic diagram of another optional implementation of step S120.

[0031] FIG29 is a schematic diagram of the degradation process of the dermal acellular matrix patch provided in Example 1.

[0032] FIG30 is a schematic diagram of the degradation process of the dermal acellular matrix patch provided in Example 3.

[0033] FIG31 is a schematic diagram of the dermal acellular matrix patch provided in the present application. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0035] References in this specification to "one embodiment," "an example," or "an example" mean that a particular feature, structure, or characteristic described in connection with the embodiment itself can be included in at least one embodiment disclosed herein. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment.

[0036] The “spinal direction” described in this application has the same meaning as commonly understood by those skilled in the art to which this application pertains.

[0037] The acellular dermal matrix (ADM) has a natural collagen fiber network structure with excellent mechanical properties. This collagen network is a cell-friendly structure, and when implanted in the body, it can promote the repair of damaged tissue. However, ADM is susceptible to degradation in the body, and it is possible that the collagen material of the ADM has completely degraded, but the damage has not yet been repaired.

[0038] Appropriate cross-linking of the collagen material of the dermal acellular matrix can prolong its degradation time. However, cross-linking of the collagen network will reduce collagen activity, thereby prolonging the healing time.

[0039] In view of this, as shown in FIG31 , as a first aspect of the present application, a dermal acellular matrix patch A is provided, wherein the dermal acellular matrix patch A includes a plurality of patch regions 10, and the plurality of patch regions 10 have at least two cross-linking properties.

[0040] Patch regions with different cross-linking properties have correspondingly different degradation times. Patch regions with shorter degradation times have relatively higher collagen activity, while patch regions with longer degradation times have relatively lower collagen activity. When the acellular dermal matrix is ​​implanted in vivo to repair damaged tissue, patch regions with higher collagen activity can repair the damaged area more quickly, while patch regions with lower collagen activity remain in the body longer, enabling comprehensive repair of the damaged tissue.

[0041] In the embodiments of the present application, the cross-linking properties of each patch area of ​​the dermal acellular matrix patch can be designed according to the specific application scenario.

[0042] In the present application, by providing a patch region 10 having at least two cross-linking properties, patch regions with different collagen activities can be organically combined, so that the dermal acellular matrix patch has a continuous and stable repair effect.

[0043] In the embodiments of the present application, there is no particular limitation on the cross-linking characteristics, as long as different cross-linking characteristics correspond to different collagen activities. In some embodiments, the cross-linking characteristics include at least one of the degree of cross-linking, the type of cross-linking network, and the cross-linking agent.

[0044] The cross-linking characteristics can be controlled by controlling the process parameters of the cross-linking process (including cross-linking agent concentration, time, temperature, pH value, etc.) and the type of cross-linking agent used in the cross-linking process.

[0045] The collagen network of at least one region of a non-crosslinked dermal acellular matrix patch (hereinafter referred to as a base patch for ease of description) can be crosslinked by applying a crosslinking agent to the patch and placing the patch under corresponding crosslinking reaction conditions.

[0046] For cross-linked networks with different cross-linking degrees obtained by the same cross-linking agent, the higher the cross-linking degree, the longer the degradation time.

[0047] For the same crosslinker network, the dermal acellular matrix patch can be made to have different degrees of crosslinking by adjusting the crosslinker concentration, crosslinking time, and crosslinking temperature. For example, the area without crosslinker added is the uncrosslinked patch area, the area treated with low crosslinker concentration and / or short crosslinking time is the low-crosslinked patch area, the area treated with medium crosslinker concentration and / or medium crosslinking time is the medium-crosslinked patch area, and the area treated with high crosslinker concentration and / or long crosslinking time is the high-crosslinked patch area.

[0048] During cross-linking, cross-linking agents introduce covalent bonds within and between collagen molecules, forming a cross-linked network. Different cross-linking agents produce different cross-linked networks, and their degradation time may also vary.

[0049] In some embodiments, different functional group pairs between collagen molecules form different cross-linked networks.

[0050] In the embodiments of the present application, the specific locations and structures of the multiple patch regions are not particularly limited, as long as the acellular dermal matrix patch has at least two different cross-linking properties. For example, the patch regions can be strip-shaped regions (e.g., rectangular or curved) or block-shaped regions.

[0051] In some embodiments, at least one of the plurality of patch regions is an uncrosslinked patch region. The uncrosslinked patch region has good collagen activity, degrades faster during tissue healing, and provides space for early tissue ingrowth.

[0052] In some embodiments, as shown in Figures 1, 5, 9, and 13, the plurality of patch regions include a central region and a peripheral region 100 disposed around the central region, wherein the central region includes at least one first central region 210 and at least one second central region 220. It is readily understood that the cross-linking properties of at least two of the peripheral region 100, the first central region 210, and the second central region 220 are different from each other.

[0053] In the embodiment of the present application, there is no particular limitation on the specific shapes of the first central area 210 and the second central area 220. For example, the first central area 210 and the second central area 220 can be selected from any one of a strip area and a block area.

[0054] In the embodiment shown in FIG1 , the first central region 210 and the second central region 220 are both strip-shaped regions. Of course, the strip-shaped regions can be rectangular (as shown in FIG1 ) or curved (as shown in FIG5 ), which is not limited in the present embodiment.

[0055] In some embodiments, the cross-linking characteristics of the first central region 210 are different from the cross-linking characteristics of the peripheral region 100 , and the cross-linking characteristics of the second central region 220 are the same as the cross-linking characteristics of the peripheral region 100 .

[0056] As described above, the cross-linking characteristics may include the degree of cross-linking. The phrase “the cross-linking characteristics of the first central region 210 are different from the cross-linking characteristics of the second central region 220 ” mentioned herein may mean that the degree of cross-linking of the first central region 210 is different from the degree of cross-linking of the second central region 220 .

[0057] It is also pointed out above that the cross-linking characteristics may include a cross-linking agent. The "cross-linking characteristics of the first central region 210 are different from the cross-linking characteristics of the second central region 220" mentioned here may mean that the cross-linking network of the first central region 210 is different from the cross-linking agent of the second central region 220.

[0058] In the embodiment of the present application, the first central area 210 and the second central area 220 can be arranged in parallel or crosswise. The relative position relationship between the first central area 210 and the second central area 220 can be determined according to the specific application scenario.

[0059] For example, in the embodiment shown in FIG1 , the length direction of the first central region 210 is consistent with the length direction of the second central region 220, that is, the length direction of the first central region 210 is parallel to the length direction of the second central region 220. Furthermore, in the embodiment shown in FIG1 , the acellular dermal matrix patch includes a plurality of first central regions 210 and a plurality of second central regions 220, and the first central regions 210 and the second central regions 220 are alternately arranged.

[0060] The length direction of the first central region 210 and the length direction of the second central region 220 are indicated by arrows in FIG. 1 .

[0061] In some embodiments, as shown in Figure 2, the central area includes multiple first central areas 210 and multiple second central areas 220, and the length direction of the first central area 210 is inconsistent with the length direction of the second central area 220, so that the first central area 210 and the second central area 220 intersect with each other and define a cell 230.

[0062] The structural design of defining the cells 230 by intersecting the first central area 210 and the second central area 220 helps to improve the mechanical strength of the patch.

[0063] The cross-linking characteristics of the first central area 210 and the second central area 220 , as well as the cells 230 and the peripheral area 100 may be set according to specific application scenarios.

[0064] In this application, taking Figure 1 as an example, the length direction of the first central region 210 and the length direction of the second central region 220 are indicated by arrows in Figure 1. Taking Figure 2 as an example, the length direction of the first central region 210 is indicated by the x direction in Figure 2, and the length direction of the second central region 220 is indicated by the y direction in Figure 2.

[0065] In the embodiments shown in Figures 2 and 10 , the crosslinking properties of the first central region 210 are the same as those of the second central region 220. The crosslinking properties of the first central region 210 and the second central region 220 are different from the crosslinking properties of the cell 230, and the crosslinking properties of the cell 230 are the same as those of the peripheral region 100. In the embodiments shown in Figures 2 and 6 , the first central region 210 and the second central region 220 are moderately crosslinked patch regions, while the cell 230 and the peripheral region 100 are uncrosslinked patch regions. In the embodiment shown in Figure 10 , the first central region 210 and the second central region 220 are moderately crosslinked patch regions, while the cell 230 and the peripheral region 100 are lightly crosslinked patch regions.

[0066] Of course, the embodiments of the present application are not limited to this. In some embodiments, the crosslinking characteristics of the first central region 210, the crosslinking characteristics of the second central region 220, and the crosslinking characteristics of the unit cells 230 are different from each other, and the crosslinking characteristics of the unit cells 230 are the same as the crosslinking characteristics of the peripheral region 100. In the embodiments shown in Figures 3, 4, 7, and 8, the first central region 210 is a moderately crosslinked patch region, the second central region 220 is a highly crosslinked patch region, and the peripheral region 100 and the unit cells 230 are uncrosslinked patch regions. In the embodiments shown in Figures 11, 12, 15, and 16, the first central region 210 is a moderately crosslinked patch region, the second central region 220 is a highly crosslinked patch region, and the peripheral region 100 and the unit cells 230 are lightly crosslinked patch regions.

[0067] In view of the above situation, the cross-linking degree may satisfy the following relationship: cross-linking degree of the first central region ≥ cross-linking degree of the second central region > cross-linking degree of the unit cell = cross-linking degree of the peripheral region.

[0068] Of course, the embodiments of the present application are not limited thereto. For example, the crosslinking networks of the first central region 210 and the second central region 220 may be different from each other, the crosslinking agent of the cell 230 may be the same as the crosslinking agent of the peripheral region 100, and the crosslinking agent of the first central region 210 and the crosslinking agent of the second central region 220 may be different from the crosslinking agent of the peripheral region 100.

[0069] In the embodiment of the present application, there is no special limitation on the specific size of the dermal acellular matrix patch, the size of the first central area 210, and the size of the second central area 220. The sizes can be set according to the actual application scenario.

[0070] In some embodiments, the width of the first central region is between 0.1 mm and 10 mm, and in some embodiments, the width of the first central region is between 0.1 mm and 5 mm. In some embodiments, the width of the second central region is between 0.1 mm and 10 mm, and in some embodiments, the width of the first central region is between 0.1 mm and 5 mm.

[0071] What is described above is a dermal acellular matrix patch including a middle region and a peripheral region 100 , but the embodiments of the present application are not limited thereto.

[0072] In the embodiment shown in Figure 17, the multiple patch areas of the dermal acellular matrix patch include at least one first patch area 310 and at least one second patch area 320, the first patch area 310 and the second patch area 320 are arranged alternately in parallel, and the cross-linking degree of the first patch area 310 is different from the cross-linking degree of the second patch area 320.

[0073] In the above embodiment, the multiple patch regions of the acellular dermal matrix patch are divided into two types. However, the present invention is not limited thereto. For example, the multiple patch regions may include a first patch region, a second patch region, a third patch region, a fourth patch region, and so on, which are arranged in parallel and in sequence, and the crosslinking properties of the multiple patch regions are different.

[0074] In some embodiments, as shown in Figure 18, the multiple patch areas include multiple first patch areas 310, multiple second patch areas 320 and multiple cell areas 330, the multiple first patch areas 310 and the multiple second patch areas 320 intersect with each other and define multiple cell areas 330, and the cross-linking degree of the first patch area 310, the cross-linking degree of the second patch area 320 and the cross-linking degree of the cell area 330 are different from each other.

[0075] In the embodiment of the present application, the first patch region 310 is a low-crosslinked patch region, the second patch region is a high-crosslinked patch region, and the cell 330 is an uncrosslinked patch region. It should be noted that the terms "high" and "low" here refer to relative crosslinking degrees.

[0076] During the early stages of implantation, the dermal acellular matrix patch provides relatively consistent support in all directions. Following implantation, as the uncrosslinked patch region (i.e., cells 330), the lowly crosslinked patch region (i.e., first patch region 310), and the highly crosslinked patch region (i.e., second patch region 320) degrade in sequence, the dermal acellular matrix patch exhibits anisotropic mechanical strength support, thereby adapting to the tissue stress of the tissue into which the dermal acellular matrix patch is implanted, better matching the healing of the tissue at different stages and in different directions.

[0077] This type of dermal acellular matrix can provide differentiated mechanical strength support in all directions in the early stages of degradation, making it particularly suitable for repairing severely damaged tendon tissue. The dermal acellular matrix is ​​implanted into the damaged tendon area along the length of the second patch region 320, parallel to the tendon bundle. The highly cross-linked second patch region has higher mechanical strength and longer degradation time, and after implantation, it can provide longer-term mechanical support along the tendon bundle direction; the low-cross-linked region can share tendon tension in another direction, preventing tendon tearing in this direction; and the network-like differentiated cross-linked network can gradually exhibit differentiated mechanical compliance properties, adapt to tissue stress, undergo appropriate deformation, and better match tissue healing.

[0078] In the embodiment shown in Figure 18, the second patch region 320 is a rectangular strip, while in the embodiment shown in Figure 19, the second patch region 320 is a curved strip. The curved strip-shaped second patch region can withstand certain deformation and stress in the direction of the curve, while also providing certain mechanical reinforcement in directions perpendicular to the curve. The embodiment shown in Figure 19 can provide differentiated mechanical strength support in various directions during early implantation of damaged tissue, making it particularly suitable for repairing damaged tissue that requires certain stress and shape change. During repair, the highly cross-linked second patch region 320 is implanted into the damaged area with its length parallel to the supraspinatus tendon fascicle. The second patch region 320 has relatively high mechanical strength and a longer degradation time. After implantation, it can provide a wider range of dynamic mechanical matching and longer-term dynamic mechanical support along the tendon fascicle to cope with dynamic tension generated during tissue repair and dynamic mechanical stimulation from external factors. The first patch region 310 is a low-cross-linked region that can share tendon tension in another direction, preventing further tendon tearing in this direction.

[0079] After the dermal acellular matrix patch is implanted, the non-crosslinked patch area (i.e., cells 330) and the low-crosslinked patch area (i.e., the first patch area 310) are degraded in sequence, and the new tissue replaces the degraded part of the dermal acellular matrix patch to achieve tissue repair. Since the undegraded highly crosslinked patch area (i.e., the second patch area 320) is distributed in a curved group, it can withstand certain deformation and stress in the direction of the curve extension. The new tissue in the repair area grows around the highly crosslinked patch area and can also withstand certain deformation and stress along with the highly crosslinked area. In addition, the new tissue formed by this network distribution is regularly arranged in multiple directions and has a larger contact area with the highly crosslinked patch area, which makes the repair area have better tear resistance.

[0080] In the embodiment shown in FIG20 , the crosslinking agents used in the first patch region 310 and the second patch region 320 are different. In other words, the crosslinking agents used in the first patch region 310 and the second patch region 320 may be different. In some embodiments, the crosslinking agent used in the first patch region 310 is 1,4-butanediol diglycidyl ether, and the crosslinking agent used in the second patch region 320 is glutaraldehyde.

[0081] 20 , the cross-linked networks of the first patch region 310 and the second patch region 320 are different in type. That is, the cross-linked networks formed by the first patch region 310 and the second patch region 320 connect different pairs of functional groups from the decellularized matrix.

[0082] 21 , the first patch region 310 and the second patch region 320 use the same crosslinking agent, which is different from the crosslinking agent used in the unit cell 330. Although the first patch region 310 and the second patch region 320 use the same crosslinking agent, in some embodiments, the degree of crosslinking in the first patch region 310 is lower than that in the second patch region 320.

[0083] For ease of description, the crosslinking agent in the first and second patch regions 310 and 320 is referred to as the first crosslinking agent, and the crosslinking agent in the unit cell 330 is referred to as the second crosslinking agent. The crosslinked network formed by the second crosslinking agent degrades faster than the crosslinked network formed by the first crosslinking agent.

[0084] After the dermal acellular matrix patch shown in FIG21 is implanted into the damaged tissue, due to the relatively high cross-linking network strength of the cell 330, the dermal acellular matrix patch can provide relatively uniform mechanical strength support in all directions in the early stages of implantation. The cross-linking network of the cell 330 degrades faster than the cross-linking network of the first patch region 310 and the cross-linking network of the second patch region 320. After implantation, the cross-linking network of the cell 330, the cross-linking network of the first patch region 310, and the cross-linking network of the second patch region 320 degrade in sequence, causing the dermal acellular matrix patch to gradually exhibit uniform mechanical strength support to anisotropic mechanical strength support after implantation, which can adapt to the tissue stress at the implantation site and better match the healing of tissues at different stages and in different directions over a long period of time.

[0085] In the embodiment shown in Figure 22, the first patch region 310 and the second patch region 320 use the same crosslinking agent, which is different from the crosslinking agent used in the unit cell 330. Although the first patch region 310 and the second patch region 320 use the same crosslinking agent, in some embodiments, the degree of crosslinking in the first patch region 310 is lower than that in the second patch region 320. In addition, the second patch region 320 is shaped like a curved bar.

[0086] The second patch area 320 with a high degree of cross-linking can withstand certain deformation and strain in the direction of the curve extension, and at the same time provide certain mechanical enhancement in the direction perpendicular to the curve. The second patch area 320 in the shape of a curved strip has relatively high mechanical strength and a relatively long degradation time. After implantation, it can provide a wider range of dynamic mechanical matching and longer-term dynamic mechanical support in a direction parallel to the length direction of the second patch area 320 to cope with the dynamic tension generated during tissue repair and the dynamic mechanical stimulation brought by the outside world. The first patch area 310 can share the tendon tension in another direction to avoid further tearing of the tendon in this direction. After the dermal acellular matrix is ​​implanted, the cells 330 and the first patch area 310 are degraded in turn, and the new tissue replaces the corresponding degradation area for tissue repair. The second patch area 320 is distributed in a curve group and can withstand certain deformation and stress in the direction of the curve extension. The new tissue in the repair area grows around the second patch area 320 and can also withstand certain deformation and stress along with the second patch area 320; at the same time, the new tissue formed by this network distribution is regularly arranged in multiple directions and has a larger contact area with the second patch area 320 with a high degree of cross-linking, so that the repair area has better tear resistance.

[0087] 23 , the dermal acellular matrix patch includes a first patch region 310, a second patch region 320, and a plurality of cells 330 defined by the first patch region 310 and the second patch region 320. In this embodiment, the crosslinking agents of the first patch region 310, the second patch region 320, and the cells 330 are different from each other.

[0088] In some embodiments, the crosslinking agent of the first patch region 310 is glutaraldehyde, the crosslinking agent of the second patch region 320 is 1,4-butanediol diglycidyl ether, and the crosslinking agent of the unit cell 330 is 1,4-butanediol diglycidyl ether.

[0089] In the examples of this application, the specific type of crosslinking agent used is not particularly limited. In some embodiments, the crosslinking agent used in the region of the acellular dermal matrix patch where the degree of crosslinking is greater than 0 is selected from at least one of the following crosslinking agents: aldehyde crosslinkers, carbodiimide, genipin, and epoxy crosslinkers.

[0090] In some embodiments, the crosslinking agent used is an epoxy crosslinking agent.

[0091] In some embodiments, the epoxy crosslinking agent may be 1,4-butanediol diglycidyl ether, and the aldehyde crosslinking agent may be glutaraldehyde.

[0092] It should be noted that although the embodiment of the present invention only shows the case where the edge is a wavy line, the embodiment of the present invention is not limited thereto. For example, the edge can be an arc, a zigzag, a broken line, or other curved lines.

[0093] As a second aspect of an embodiment of the present application, a method for preparing a dermal acellular matrix patch is provided, wherein, as shown in FIG24 , the preparation method includes the following steps.

[0094] In step S110 , an initial dermal acellular matrix patch is provided.

[0095] In step S120, the initial dermal acellular matrix patch is pattern-crosslinked to obtain the dermal acellular matrix patch, wherein the dermal acellular matrix patch includes a plurality of patch regions, and the plurality of patch regions have at least two crosslinking properties.

[0096] The preparation method provided in the embodiment of the present application can be used to prepare the dermal acellular matrix patch provided in the first aspect of the present application. The working principle and beneficial effects of the dermal acellular matrix patch have been described in detail above and will not be repeated here.

[0097] In the present embodiment, there is no particular limitation on how to perform "patterned crosslinking." For example, a crosslinking agent can be sprayed only on the initial dermal acellular matrix patch by inkjet printing, ultimately obtaining a dermal acellular matrix patch comprising multiple patch regions.

[0098] Of course, the embodiments of the present application are not limited thereto. To reduce preparation costs and facilitate production, in some embodiments, as shown in FIG25 , the patterned cross-linking of the initial dermal acellular matrix patch includes the following steps.

[0099] In step S121, a patterning tool is provided on the surface of the initial dermal acellular matrix patch, wherein the patterning tool includes at least one shielding area and at least one hollow area.

[0100] In step S122, the initial acellular dermal matrix patch, equipped with the patterning tool, is cross-linked using a first cross-linking agent. The hollowed-out areas are exposed to the first cross-linking agent, allowing a cross-linking reaction to occur. The patterning tool can be a mask or a fixture. To facilitate the cross-linking reaction, in some embodiments, the patterning tool is a fixture. The fixture is used to hold the patch and then place it in the cross-linking agent for cross-linking.

[0101] In some embodiments, the patch after step S122 can be directly used as the final dermal acellular matrix patch. In some embodiments, as shown in Figures 26 and 27, the shielding area 120 and the hollow area 200 are both strip-shaped, and the patterning tool includes multiple shielding areas 120 and multiple hollow areas 200. The length direction of the shielding area 120 is consistent with the length direction of the hollow area 200, and the shielding areas 120 and the hollow areas 200 are alternately arranged.

[0102] For ease of operation, the patterning tool may further include a frame 110, which is disposed around the shielding area 120 and the hollow area 200. Using the patterning tool shown in FIG26 , the dermal acellular matrix patch shown in FIG17 can be obtained. It should be noted that if the initial dermal acellular matrix patch extends beyond the central region where the shielding area 120 and the hollow area 200 are located, the edge of the initial dermal acellular matrix patch will be shielded by the frame 110, and the shielded portion will not undergo a cross-linking reaction, thereby obtaining the dermal acellular matrix patch shown in FIG1 .

[0103] In the embodiment shown in Figure 27 , the edges of the shielding area 120 and the hollow area 200 are curved. The dermal acellular matrix patch shown in Figure 5 can be obtained by using the patterning tool shown in Figure 27 .

[0104] In some embodiments, the patch obtained after step S122 is used as an intermediate product and further processed. In other words, the patterned cross-linking of the initial dermal acellular matrix patch may further include the following steps.

[0105] In step S123, the patterning tool is removed to obtain an intermediate patch.

[0106] In step S124, the middle patch piece is cross-linked using a second cross-linking agent.

[0107] In the embodiment of the present application, the second cross-linking agent can be the same as or different from the first cross-linking agent. When the second cross-linking agent is the same as the first cross-linking agent, while the first patch region 310 undergoes a cross-linking reaction, the second patch region 320 also undergoes a further cross-linking reaction. In the resulting dermal acellular matrix patch, the degree of cross-linking in the second patch region 320 is higher than that in the first patch region 310, as shown in FIG20 . When the edge of the initial dermal acellular matrix patch reaches the border of the patterning tool, the dermal acellular matrix patch shown in FIG9 or FIG11 can be obtained through steps S121 to S124.

[0108] As another optional embodiment of the present application, as shown in FIG28 , the patterned cross-linking of the initial dermal acellular matrix patch to obtain the dermal acellular matrix patch includes the following steps.

[0109] In step S125, a first patterning tool is set on the surface of the initial dermal acellular matrix patch, and the first patterning tool includes at least one first shielding area and at least one first hollow area, the length direction of the first shielding area is consistent with the length direction of the first hollow area, and the first shielding area and the first hollow area are alternately arranged.

[0110] In step S126, the initial dermal acellular matrix patch provided with the first patterning tool is cross-linked using a first cross-linking agent. In step S127, the first patterning tool is removed to obtain a first intermediate patch. In step S128, a second patterning tool is provided on the surface of the first intermediate patch. The second patterning tool includes at least one second shielding area and at least one second hollow area. The length direction of the second shielding area is consistent with the length direction of the second hollow area. The second shielding areas and the second hollow areas are alternately provided, and the length direction of the second shielding areas intersects with the length direction of the first shielding areas.

[0111] In step S129 , the first intermediate patch provided with the second patterning tool is cross-linked using a second cross-linking agent.

[0112] In the embodiment of the present application, the first patterning tool and the second patterning tool can be the same fixture or different fixtures. The patch obtained after step S129 can be the final dermal acellular matrix patch (as shown in Figures 2, 3, 4, 6, 7, 8, 18, and 19).

[0113] In the embodiments of the present application, there is no particular limitation on the specific types of the first cross-linking agent and the second cross-linking agent. The first cross-linking agent can be the same as the second cross-linking agent or different from the second cross-linking agent.

[0114] As another optional embodiment of the present application, as shown in FIG28 , the patterned cross-linking of the initial dermal acellular matrix patch to obtain the dermal acellular matrix patch further includes the following steps.

[0115] In step S1210 , the second patterning tool is removed to obtain a second intermediate patch.

[0116] In step S1211 , the second intermediate patch is cross-linked using a third cross-linking agent.

[0117] Through steps S1210 and S1211, the patch obtained in step S129 can be further cross-linked to obtain dermal acellular matrix patches such as those shown in Figures 10, 11, 12, 14, 15, 16, 21, 22, and 23.

[0118] The third cross-linking agent may be different from the first cross-linking agent and the second cross-linking agent, or may be the same as the first cross-linking agent or the second cross-linking agent.

[0119] As described above, the cross-linking agent is selected from at least one of the following cross-linking agents: aldehyde cross-linking agent, carbodiimide, genipin, and epoxy cross-linking agent.

[0120] As a third aspect of the present application, a use of a dermal acellular matrix patch in repairing tissue damage is provided, wherein the dermal acellular matrix patch is the dermal acellular matrix patch described in the first aspect of the present application.

[0121] Example 1

[0122] The dermal acellular matrix patch shown in FIG17 was prepared using the following preparation method.

[0123] A freeze-dried initial dermal acellular matrix patch was taken and clamped with a linear strip grid clamp (i.e., patterned tool) and immersed in a 2% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 11-14 using 5M NaOH and cross-linked at 30°C for 12 hours.

[0124] The cross-linked dermal acellular matrix patch was rinsed with plenty of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried to obtain the dermal acellular matrix patch shown in FIG17 .

[0125] Gradient degradation of Example 1

[0126] The surface gradient patterned cross-linked patch prepared according to the above process (as shown in FIG17 ) was added to a 1% pancreatic enzyme solution and soaked at 37° C. to verify the degradation performance of the patch. The degradation of the patch was observed after 24 h and 48 h.

[0127] As shown in Figure 29, after immersion in 1% pancreatic enzyme solution for 1 hour, yellowish stripes were observed in the hollowed-out area (cross-linked surface), while obvious whitish swelling was observed in the blocked area (non-cross-linked surface). After immersion in 1% pancreatic enzyme solution for 24 hours, the uncross-linked area began to degrade, while the cross-linked area remained unchanged. After immersion for 48 hours, the uncross-linked area had almost disappeared, leaving only the strip-like cross-linked area in contact with the paste. This phenomenon indicates that the patterned cross-linking of the acellular dermal matrix patch produced a distinct patterned effect of cross-linked and uncross-linked areas.

[0128] Example 2

[0129] The dermal acellular matrix patch shown in FIG18 was prepared using the following preparation method.

[0130] A freeze-dried initial dermal acellular matrix patch was taken and clamped with a vertical striped grid clamp. The patch was then immersed in a 0.5% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 12 with 5 M NaOH and cross-linked at 30°C for 5 h.

[0131] The cross-linked dermal acellular matrix patch (middle patch) was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0132] After the middle patch was freeze-dried, the vertical striped grid clamp was reused to clamp the middle patch in a direction perpendicular to the direction of clamping the initial dermal acellular matrix patch. The middle patch was immersed in a 2% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 11-14 using 5M NaOH and cross-linked at 50°C for 12 hours.

[0133] The gridded cross-linked dermal acellular matrix patch (as shown in FIG18 ) was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0134] Example 3

[0135] The dermal acellular matrix patch shown in FIG19 was prepared using the following preparation method.

[0136] A freeze-dried initial dermal acellular matrix patch was taken and clamped with a vertical striped grid clamp. The matrix was immersed in a 0.5% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 12 with 5M NaOH and cross-linked at 30°C for 5 hours.

[0137] The cross-linked middle patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0138] The freeze-dried intermediate patch was re-clamped with a curved strip-shaped grid clamp and immersed in a 2% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 11-14 using 5M NaOH and cross-linked at 50°C for 12 hours. The length direction of the curved strip was perpendicular to the strip direction of the vertical strip-shaped grid clamp that clamped the initial dermal acellular matrix patch.

[0139] The gridded cross-linked dermal acellular matrix patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0140] Degradation of Example 3

[0141] As shown in Figure 30 , a cross-linked dermal acellular matrix patch with a gradient patterned surface, prepared according to the above process, was immersed in a 1% pancreatic enzyme solution at 37°C to verify the degradation performance of the patch. Degradation of the patch was observed after 24 hours. Figure 30A shows the dermal acellular matrix patch before cutting, Figure 30B shows the patch before degradation, and Figure 30C shows the degradation of the patch after 24 hours.

[0142] In Example 3, highly cross-linked regions of the curved strips are clearly observed. After immersion in a 1% trypsin solution for 24 hours, the uncross-linked regions in Example 3 have become completely transparent, while the highly cross-linked curved regions show little degradation, and the curved strips are not completely dispersed. This may be due to the presence of localized low-cross-linked regions that fix the highly cross-linked curved regions. This phenomenon demonstrates that patterned cross-linking can be used to prepare a decellularized matrix, creating a distinct grid pattern of cross-linked and uncross-linked regions.

[0143] Example 4

[0144] The dermal acellular matrix patch shown in FIG20 was prepared using the following preparation method.

[0145] A freeze-dried initial dermal acellular matrix patch was taken and clamped with a vertical striped grid clamp. The matrix was immersed in a 2% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 11-14 with 5M NaOH and cross-linked at 30°C for 12 hours to obtain an intermediate patch.

[0146] The middle patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0147] The freeze-dried middle patch was immersed in 0.5% (w / w) glutaraldehyde solution and reacted at 30°C for 1 hour. The reacted middle patch was rinsed with plenty of water, soaked in 0.5% SDS solution for swelling for 1 hour and then freeze-dried to obtain the dermal acellular matrix patch.

[0148] Example 5

[0149] The dermal acellular matrix patch shown in FIG21 was prepared using the following preparation method.

[0150] A freeze-dried initial dermal acellular matrix patch was taken, clamped with a vertical striped grid clamp, and immersed in a 0.5% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 12 with 5M NaOH, and cross-linked at 30°C for 5 hours to obtain the first intermediate patch.

[0151] The first middle patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0152] The freeze-dried first intermediate patch was re-clamped with a vertical striped grid clamp in a direction perpendicular to the strips of the clamp holding the initial dermal acellular matrix patch, and immersed in a 2% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 11-14 using 5M NaOH, and cross-linked at 50°C for 12 hours to obtain a second intermediate patch.

[0153] The second middle patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0154] The freeze-dried second intermediate patch was immersed in 0.5% (w / w) glutaraldehyde solution and reacted at 30°C for 1 hour. The reacted acellular matrix was rinsed with a large amount of water, soaked in 0.5% SDS solution for swelling for 1 hour, and then freeze-dried to obtain the dermal acellular matrix patch.

[0155] Example 6

[0156] The dermal acellular matrix patch shown in FIG22 was prepared using the following preparation method.

[0157] A freeze-dried initial dermal acellular matrix patch was taken and clamped with a vertical striped grid clamp. The initial dermal acellular matrix patch was immersed in a 0.5% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 12 with 5M NaOH and cross-linked at 30°C for 5 hours to obtain the first intermediate patch.

[0158] The first middle patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0159] The freeze-dried first intermediate patch was re-clamped perpendicularly to the strips of the curved strip grid clamp and the clamp holding the initial dermal acellular matrix patch, and immersed in a 2% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 11-14 using 5M NaOH, and cross-linked at 50°C for 12 hours to obtain the second intermediate patch.

[0160] The second middle patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0161] The freeze-dried second intermediate patch was immersed in 0.5% (w / w) glutaraldehyde solution and reacted at 30°C for 1 hour. The reacted acellular matrix was rinsed with a large amount of water, soaked in 0.5% SDS solution for swelling for 1 hour, and then freeze-dried to obtain the dermal acellular matrix patch.

[0162] Example 7

[0163] The dermal acellular matrix patch shown in FIG23 was prepared using the following preparation method.

[0164] A freeze-dried initial dermal acellular matrix patch was taken and clamped with a vertical striped grid clamp. The matrix was immersed in 0.5% (w / w) glutaraldehyde solution and reacted at 30°C for 5 hours to obtain a first intermediate patch.

[0165] The first middle patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0166] The freeze-dried first intermediate patch was re-clamped with a vertical striped grid clamp in a direction perpendicular to the direction in which the vertical striped grid clamp clamped the initial dermal acellular matrix patch strips, and immersed in a 2% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 11-14 using 5M NaOH, and cross-linked at 50°C for 12 hours to obtain a second intermediate patch.

[0167] The second middle patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0168] The freeze-dried patch was immersed in 0.8% EDC solution and activated by adding 0.2% NHS. The pH was adjusted to 5.5 and then cross-linked at 10°C for 5 hours. The reacted acellular matrix was rinsed with a large amount of water, soaked in 0.5% SDS solution for swelling for 1 hour, and then freeze-dried to obtain the dermal acellular matrix patch.

[0169] Example 8

[0170] The following preparation method can also be used to prepare the dermal acellular matrix patch shown in FIG20 .

[0171] A freeze-dried initial dermal acellular matrix patch was taken and clamped with a vertical striped grid clamp. The matrix was immersed in a 2% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 11-14 with 5M NaOH and cross-linked at 30°C for 12 hours to obtain an intermediate patch.

[0172] The middle patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0173] The freeze-dried middle patch was immersed in a 5% (w / w) 1,4-butanediol diglycidyl ether solution, and the pH of the solution was adjusted to 8-10 using 5M NaOH. The reaction was carried out at 30°C for 48 hours. The middle patch after the reaction was rinsed with a large amount of water, immersed in a 0.5% SDS solution to swell for 1 hour, and then freeze-dried to obtain a dermal acellular matrix patch.

[0174] Example 9

[0175] The dermal acellular matrix patch shown in FIG17 was prepared using the following preparation method.

[0176] A freeze-dried initial dermal acellular matrix patch was taken and clamped with a linear strip grid clamp (i.e., a patterned tool) and immersed in a 1.5% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 8-10 using 5M NaOH and cross-linked at 30°C for 48 hours.

[0177] The cross-linked dermal acellular matrix patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried to obtain a dermal acellular matrix patch.

[0178] Example 10

[0179] The dermal acellular matrix patch shown in FIG18 was prepared using the following preparation method.

[0180] A freeze-dried initial dermal acellular matrix patch was taken and clamped with a vertical striped grid clamp. The patch was then immersed in a 0.5% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 12 with 5 M NaOH and cross-linked at 30°C for 5 h.

[0181] The cross-linked dermal acellular matrix patch (middle patch) was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0182] After the middle patch was freeze-dried, the vertical striped grid clamp was reused to clamp the middle patch in a direction perpendicular to the direction of clamping the initial dermal acellular matrix patch. The middle patch was immersed in a 2% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 8-10 using 5M NaOH and cross-linked at 50°C for 24 hours.

[0183] The gridded cross-linked dermal acellular matrix patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0184] Example 11

[0185] The dermal acellular matrix patch shown in FIG19 was prepared using the following preparation method.

[0186] A freeze-dried initial dermal acellular matrix patch was taken and clamped with a vertical striped grid clamp. The matrix was immersed in a 0.5% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 12 with 5M NaOH and cross-linked at 30°C for 5 hours.

[0187] The cross-linked middle patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0188] The freeze-dried intermediate patch was re-clamped with a curved strip-shaped grid clamp and immersed in a 5% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 8-10 using 5M NaOH and cross-linked at 30°C for 48 hours. The length direction of the curved strip was perpendicular to the direction of the vertical strip-shaped grid clamp that clamped the initial dermal acellular matrix patch.

[0189] The gridded cross-linked dermal acellular matrix patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0190] Example 12

[0191] The dermal acellular matrix patch shown in FIG20 was prepared using the following preparation method.

[0192] A freeze-dried initial dermal acellular matrix patch was taken and clamped with a vertical striped grid clamp. The matrix was immersed in a 5% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 8-10 with 5M NaOH and cross-linked at 30°C for 48 hours to obtain an intermediate patch.

[0193] The middle patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0194] The freeze-dried middle patch was immersed in 0.5% (w / w) glutaraldehyde solution and reacted at 30°C for 1 hour. The reacted middle patch was rinsed with plenty of water, soaked in 0.5% SDS solution for swelling for 1 hour and then freeze-dried to obtain the dermal acellular matrix patch.

[0195] Example 13

[0196] The dermal acellular matrix patch shown in FIG21 was prepared using the following preparation method.

[0197] A freeze-dried initial dermal acellular matrix patch was taken, clamped with a vertical striped grid clamp, and immersed in a 0.5% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 12 with 5M NaOH, and cross-linked at 30°C for 5 hours to obtain the first intermediate patch.

[0198] The first middle patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0199] The freeze-dried first intermediate patch was re-clamped with a vertical striped grid clamp in a direction perpendicular to the strips of the clamp holding the initial dermal acellular matrix patch, and immersed in a 5% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 8-10 using 5M NaOH, and cross-linked at 30°C for 48 hours to obtain a second intermediate patch.

[0200] The second middle patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0201] The freeze-dried second intermediate patch was immersed in 0.1% (w / w) glutaraldehyde solution and reacted at 30°C for 1 hour. The reacted acellular matrix was rinsed with a large amount of water, soaked in 0.5% SDS solution for swelling for 1 hour, and then freeze-dried to obtain the dermal acellular matrix patch.

[0202] Example 14

[0203] The dermal acellular matrix patch shown in FIG22 was prepared using the following preparation method.

[0204] A freeze-dried initial dermal acellular matrix patch was taken and clamped with a vertical striped grid clamp. The initial dermal acellular matrix patch was immersed in a 0.5% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 12 with 5M NaOH and cross-linked at 30°C for 5 hours to obtain the first intermediate patch.

[0205] The first middle patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0206] The freeze-dried first intermediate patch was re-clamped perpendicularly to the strips of the curved strip grid clamp and the clamp holding the initial dermal acellular matrix patch, and immersed in a 5% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 8-10 using 5M NaOH, and cross-linked at 30°C for 48 hours to obtain a second intermediate patch. The second intermediate patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in a 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0207] The freeze-dried second intermediate patch was immersed in 0.5% (w / w) glutaraldehyde solution and reacted at 30°C for 1 hour. The reacted acellular matrix was rinsed with a large amount of water, soaked in 0.5% SDS solution for swelling for 1 hour, and then freeze-dried to obtain the dermal acellular matrix patch.

[0208] Example 15

[0209] The dermal acellular matrix patch shown in FIG23 was prepared using the following preparation method.

[0210] A freeze-dried initial dermal acellular matrix patch was taken and clamped with a vertical striped grid clamp. The matrix was immersed in 0.5% (w / w) glutaraldehyde solution and reacted at 30°C for 5 hours to obtain a first intermediate patch.

[0211] The first middle patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0212] The freeze-dried first intermediate patch was re-clamped with a vertical striped grid clamp in a direction perpendicular to the direction in which the vertical striped grid clamp clamped the initial dermal acellular matrix patch strips, and immersed in a 5% (w / w) solution of 1,4-butanediol diglycidyl ether. The pH of the solution was adjusted to 8-10 using 5M NaOH, and cross-linked at 30°C for 48 hours to obtain a second intermediate patch.

[0213] The second middle patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0214] The freeze-dried patch was immersed in 0.8% EDC solution and activated by adding 0.2% NHS. The pH was adjusted to 5.5 and then cross-linked at 10°C for 5 hours. The reacted acellular matrix was rinsed with a large amount of water, soaked in 0.5% SDS solution for swelling for 1 hour, and then freeze-dried to obtain the dermal acellular matrix patch.

[0215] Comparative Example 1

[0216] A freeze-dried bovine dermal acellular matrix patch was placed in a 2% 1,4-butanediol diglycidyl ether solution, the pH of the solution was adjusted to 12-13, cross-linked at 30°C for 12 hours, rinsed with plenty of water, replaced with ethanol twice, and then immersed in a 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried to obtain a dermal acellular matrix.

[0217] Comparative Example 2

[0218] A freeze-dried bovine dermal acellular matrix patch was taken as the blank control group.

[0219] Comparative Example 3

[0220] A freeze-dried bovine dermal acellular matrix patch was taken and immersed in a 0.5% (w / w) glutaraldehyde solution, and reacted at 30° C. for 5 h to obtain a first intermediate patch.

[0221] The first middle patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0222] The freeze-dried first middle patch was immersed in a 5% (w / w) solution of 1,4-butanediol diglycidyl ether, the pH of the solution was adjusted to 8-10 using 5M NaOH, and cross-linked at 30°C for 48 hours to obtain a second middle patch.

[0223] The second middle patch was rinsed with a large amount of water, replaced with ethanol twice, and then immersed in 0.5% SDS solution. After swelling for 1 hour, it was replaced with purified water and freeze-dried.

[0224] The freeze-dried patch was immersed in a 0.8% EDC solution and activated by adding 0.2% NHS. The pH was adjusted to 5.5 and then cross-linked at 10°C for 5 hours. The reacted acellular matrix was rinsed with a large amount of water, soaked in a 0.5% SDS solution to swell for 1 hour, and then freeze-dried to obtain the dermal acellular matrix patch of Comparative Example 2.

[0225] Test Case

[0226] The tensile strength test was performed on samples of Examples 1-15 and Comparative Examples 1-3 at different degradation time periods.

[0227] Degradation method: The surface gradient patterned cross-linked patch prepared according to the above process was added with 1% pancreatic enzyme solution and soaked at 37° C. for 12 h, then taken out, washed, and freeze-dried.

[0228] Tensile strength test method:

[0229] The tensile strength of the decellularized matrix was tested according to the USP 2381-2384 Bovine Acellular Dermal Matrix tensile test method. Patches prepared in Examples 1-15 and Comparative Examples 1-3 were cut into horizontal and vertical specimens measuring 5 cm x 0.5 cm in length and width, respectively. A clamp was placed 1 cm on each side of the specimen. The specimen was stretched along its length at a rate of 30 mm / min, and the maximum tensile force was recorded. The thickness of each specimen was measured using a vernier caliper, and the tensile strength was calculated. The results are shown in Table 1.

[0230] For transverse samples prepared using the dermal acellular matrix patches of Examples 1-15, the length direction of the highly cross-linked / cross-linked strips was defined as the length direction; for longitudinal samples prepared using the dermal acellular matrix patches of Examples 1-15, the length direction of the lowly cross-linked / uncross-linked region strips was defined as the length direction. For transverse samples prepared using the dermal acellular matrix patches of Comparative Examples 1-3, the length direction was defined as parallel to the vertebral direction, and the width direction was defined as perpendicular to the vertebral direction; for longitudinal samples prepared using the dermal acellular matrix patches of Comparative Examples 1-3, the length direction was defined as perpendicular to the vertebral direction, and the width direction was defined as parallel to the vertebral direction.

[0231] Among them, the tensile strength is calculated by the following formula:

[0232] Tensile strength = maximum tensile force / action area; action area = spline thickness × spline width.

[0233] Table 1

[0234] As shown in the degradation data at 0 h in Table 1, the maximum transverse / longitudinal tensile force and transverse / longitudinal tensile strength of the single-patterned cross-linked patch of Example 1 were inferior to those of the single-patterned fully cross-linked dermal acellular matrix patch of Comparative Example 1 due to the presence of some uncross-linked areas, but were significantly higher than those of the uncross-linked initial dermal acellular matrix patch of Comparative Example 2. The maximum transverse / longitudinal tensile force and transverse / longitudinal tensile strength of Examples 1-15, which were patterned cross-linked, were both greater than those of the initial dermal acellular matrix patch of Comparative Example 2, demonstrating that the patterned cross-linked structure disclosed herein can effectively enhance the mechanical properties of the dermal acellular matrix patch while retaining some collagen activity. Comparisons with Examples 1-3 reveal that the maximum tensile force and tensile strength of Examples 1-3 gradually increase, indicating that the provision of first and second central regions in different directions helps improve the mechanical strength of the patch. Examples 4-14 use a double or triple cross-linked network, and their maximum transverse / longitudinal tensile force and transverse / longitudinal tensile strength are similar to or even greater than the fully cross-linked patch of Comparative Example 1, further demonstrating that the patterned cross-linked structure of the present application can effectively enhance the mechanical properties of the dermal acellular matrix patch.

[0235] The dermal acellular matrix patches in Example 15 and Comparative Example 3 were both cross-linked three times. Compared to the uncross-linked dermal acellular matrix patch in Comparative Example 2, the mechanical strength of both patches was significantly improved. Further comparison of Example 15 and Comparative Example 3 revealed that while the cross-linking properties of the dermal acellular matrix patch in Example 15 varied across regions, its initial transverse and longitudinal maximum tensile forces and tensile strength were both higher than those of the multiply uniformly cross-linked patch in Comparative Example 3. In particular, the longitudinal maximum tensile force was significantly superior to that of Comparative Example 3, further demonstrating that the patterned cross-linked structure disclosed herein can effectively enhance the mechanical properties of dermal acellular matrix patches.

[0236] At the same time, the transverse or longitudinal data show that the mechanical properties in the direction parallel to the high-crosslinked area strips (transverse) are significantly higher than those in the direction perpendicular to the high-crosslinked area strips (longitudinal). Among them, the curved grid crosslinking has a higher longitudinal strength than the straight grid crosslinking patch in the direction perpendicular to the high-crosslinked area strips (longitudinal). This shows that the high-crosslinked areas of multiple curve groups can not only withstand stress in the direction of curve extension, but also have a mechanical reinforcement effect in the direction perpendicular to the curve. Compared with a single vertical strip, this type of dermal acellular matrix can provide differentiated mechanical strength support in all directions in the early stage of implantation, which is suitable for the repair of damaged tissues that require certain stress and shape changes.

[0237] Furthermore, after 12 hours of degradation, the patch of the present application can still maintain a certain mechanical strength, and the difference in transverse-longitudinal tensile strength of the patch remains almost unchanged, indicating that the patch can still achieve a good anisotropic mechanical reinforcement effect after degradation for a period of time, effectively promoting tissue repair and mechanical reinforcement in different directions, and matching the tissue repair needs of different parts and directions.

[0238] In the present application, by setting up a patch area including at least two cross-linking properties, the patch areas with different collagen activities can be organically combined, so that the dermal acellular matrix patch has a continuous and stable repair effect; through patterned cross-linking, the patch areas with different cross-linking properties in the dermal acellular matrix patch of the present application can be tightly combined, so that the dermal acellular matrix patch of the present application has higher strength, which can reach or even exceed the mechanical strength of the completely uniformly cross-linked dermal acellular matrix patch in the related technology.

[0239] The technical features of the above-described embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification. The above-described embodiments only express several implementation methods of the present application, and their descriptions are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. Therefore, the scope of protection of the patent application of this application shall be based on the attached claims.

Claims

1. A dermal acellular matrix patch, characterized in that: The dermal acellular matrix patch includes a plurality of patch regions, wherein the plurality of patch regions have at least two cross-linking properties.

2. The dermal acellular matrix patch according to claim 1, wherein: At least one of the plurality of patch regions is a non-cross-linked patch region.

3. The dermal acellular matrix patch according to claim 1, wherein: The plurality of patch regions include a central region and a peripheral region disposed around the central region.

4. The dermal acellular matrix patch according to claim 3, wherein: The central area includes at least one first central area and at least one second central area, and the first central area and the second central area are both strip-shaped areas; The length direction of the first central region is consistent with the length direction of the second central region, the crosslinking characteristics of the second central region are the same as the crosslinking characteristics of the peripheral region, and the crosslinking characteristics of the first central region are different from the crosslinking characteristics of the second central region; or The central area includes a plurality of first central areas and a plurality of second central areas, wherein the length direction of the first central area is inconsistent with the length direction of the second central area, so that the first central area and the second central area intersect with each other and define a unit cell; The cross-linking properties of each region of the acellular dermal matrix patch satisfy a first or second condition, wherein: the first condition is that the cross-linking properties of the first central region are the same as the cross-linking properties of the second central region and different from the cross-linking properties of the cell, and the cross-linking properties of the cell are the same as the cross-linking properties of the peripheral region; The second condition is that the crosslinking characteristics of the first central region, the crosslinking characteristics of the second central region, and the crosslinking characteristics of the unit cells are different from each other, and the crosslinking characteristics of the unit cells are the same as the crosslinking characteristics of the peripheral region.

5. The dermal acellular matrix patch according to claim 4, wherein: An edge of at least one of the first central area and the second central area is a curved line.

6. The dermal acellular matrix patch according to claim 1, wherein: The plurality of patch regions include at least one first patch region and at least one second patch region, and the first patch region and the second patch region are alternately arranged in parallel; or The plurality of patch regions include a plurality of first patch regions, a plurality of second patch regions, and a plurality of cell regions, and the plurality of first patch regions and the plurality of second patch regions intersect with each other.

7. The dermal acellular matrix patch according to claim 6, wherein: An edge of at least one of the first patch area and the second patch area is a curved line.

8. A method for preparing a dermal acellular matrix patch, wherein: The preparation method comprises: Provides an initial dermal acellular matrix patch; The initial dermal acellular matrix patch is subjected to patterned cross-linking to obtain the dermal acellular matrix patch, The dermal acellular matrix patch includes a plurality of patch regions, wherein the plurality of patch regions have at least two cross-linking properties.

9. The preparation method according to claim 8, wherein The patterned cross-linking of the initial dermal acellular matrix patch comprises: Arranging a patterning tool on the surface of the initial dermal acellular matrix patch, wherein the patterning tool comprises at least one shielding area and at least one hollow area; The initial dermal acellular matrix patch provided with the patterning tool is cross-linked using a first cross-linking agent.

10. The preparation method according to claim 9, wherein The blocking area and the hollow area are both in the shape of bars. The patterning tool includes multiple blocking areas and multiple hollow areas. The length direction of the blocking area is consistent with the length direction of the hollow area. The blocking areas and the hollow areas are alternately arranged.

11. The preparation method according to claim 10, wherein Patterning and cross-linking the initial dermal acellular matrix patch also includes: removing the patterning tool to obtain an intermediate patch; The middle patch piece is cross-linked using a second cross-linking agent.

12. The preparation method according to claim 8, wherein The initial dermal acellular matrix patch is subjected to patterned cross-linking to obtain the dermal acellular matrix patch, comprising: A first patterning tool is provided on the surface of the initial dermal acellular matrix patch, wherein the first patterning tool includes at least one first shielding area and at least one first hollowing area, wherein the length direction of the first shielding area is consistent with the length direction of the first hollowing area, and the first shielding areas and the first hollowing areas are alternately provided; cross-linking the initial dermal acellular matrix patch provided with the first patterning tool using a first cross-linking agent; removing the first patterning tool to obtain a first intermediate patch; A second patterning tool is provided on the surface of the first intermediate patch, wherein the second patterning tool includes at least one second blocking area and at least one second hollow area, wherein the length direction of the second blocking area is consistent with the length direction of the second hollow area, and the second blocking areas and the second hollow areas are alternately provided, and the length direction of the second blocking area intersects with the length direction of the first blocking area; The first intermediate patch provided with the second patterning tool is cross-linked using a second cross-linking agent.

13. The preparation method according to claim 12, wherein The method of patterning and cross-linking the initial dermal acellular matrix patch to obtain the dermal acellular matrix patch further comprises: removing the second patterning tool to obtain a second intermediate patch; The second middle patch is cross-linked using a third cross-linking agent.

14. Use of a dermal acellular matrix patch in repairing tissue damage, wherein: The dermal acellular matrix patch is the dermal acellular matrix patch according to any one of claims 1 to 8.

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