Biological patch, preparation method therefor, and use thereof

Abstract

A biological patch, a preparation method therefor, and use thereof. The biological patch comprises a substrate. In the thickness direction of the biological patch, the substrate comprises a non-cross-linked portion and a cross-linked portion, wherein the thickness ratio of the non-cross-linked portion to the cross-linked portion is 1: 99 to 99: 1; the porosity of the non-cross-linked portion is greater than the porosity of the cross-linked portion, and the porosity difference is greater than or equal to 5%.

Description

Biological patches, their preparation methods and applications

[0001] Related applications

[0002] This application claims priority to Chinese Patent Application No. 202411802393.8, filed on December 6, 2024, entitled "Biological Patch and Preparation Method and Application Thereof", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the field of medical materials technology, and in particular to biological patches, their preparation methods, and applications. Background Technology

[0004] Existing biological patches degrade too quickly after implantation. Cross-linking processes are generally used to improve the degradation performance of biological patches, but there is still a problem that the degradation rate of biological patches does not match the tissue repair process. In some cases, there is even excessive cross-linking, which reduces the bioactivity of collagen in the biological patch, prolongs the healing time, and may even lead to repair failure. Summary of the Invention

[0005] According to various embodiments of this application, a biological patch, its preparation method, and its application are provided. This biological patch can prolong its degradation time in vivo, maintain good mechanical properties, retain high collagen bioactivity, and its degradation rate in vivo matches the tissue repair process, thus better promoting the comprehensive repair of damaged tissues.

[0006] A biological patch includes a matrix. Along the thickness direction of the biological patch, the matrix includes a non-crosslinked portion and a crosslinked portion, wherein the thickness ratio of the non-crosslinked portion to the crosslinked portion is 1:99-99:1, the porosity of the non-crosslinked portion is greater than the porosity of the crosslinked portion, and the porosity difference is greater than or equal to 5%.

[0007] In one embodiment, the average porosity of the non-crosslinked portion is 40%-90%.

[0008] In one embodiment, the average porosity of the crosslinked portion is 0.1%-40%.

[0009] In one embodiment, the porosity of the cross-linked portions is the same from the direction of the non-cross-linked portions, or the porosity of the cross-linked portions increases in a gradient.

[0010] In one embodiment, when the porosity of the cross-linked portions is the same, the thickness ratio of the non-cross-linked portions to the cross-linked portions is 1:9-9:1.

[0011] In one embodiment, when the porosity of the cross-linked portion increases in a gradient, the thickness ratio of the non-cross-linked portion to the cross-linked portion is 1:99-99:1.

[0012] In one embodiment, when the porosity increases in a gradient, the cross-linked portion contains a polysaccharide polymer, and the average degree of cross-linking of the polysaccharide polymer is 0.1%-50%.

[0013] In one embodiment, the polysaccharide polymer is selected from at least one of chitosan, chitosan derivatives, cellulose, cellulose derivatives, guar gum, tamarind gum, sodium carboxymethyl cellulose, and sodium carboxymethyl starch.

[0014] In one embodiment, the non-crosslinked portion includes a first non-crosslinked portion and a second non-crosslinked portion, and the crosslinked portion is disposed between the first non-crosslinked portion and the second non-crosslinked portion. From the first non-crosslinked portion to the second non-crosslinked portion, the porosity of the crosslinked portion first decreases and then increases.

[0015] In one embodiment, the thickness ratio of the first non-crosslinked portion to the second non-crosslinked portion is 1:9-9:1.

[0016] In one embodiment, an adhesive layer is further provided between the non-crosslinked portion and the crosslinked portion, the thickness of which is 0.1%-20% of the total thickness of the substrate.

[0017] In one embodiment, the material of the matrix is ​​selected from decellularized dermal matrix or collagen.

[0018] In one embodiment, the thickness of the biological patch is 0.5 mm to 5.5 mm.

[0019] In one embodiment, the biological patch has an average pore size of 0.1 μm-500 μm and an average porosity of 10%-80%.

[0020] This application also discloses a method for preparing a biological patch, comprising the following steps: mixing an aqueous solution of a polysaccharide polymer compound and a crosslinking agent to obtain a mixed solution; placing the mixed solution on any surface of a matrix material, allowing the mixed solution to gradually penetrate into the matrix material to a preset depth to perform a gradient crosslinking reaction to form a crosslinked portion and a non-crosslinked portion; after washing and freeze-drying, a biological patch is obtained, wherein the thickness ratio of the non-crosslinked portion to the crosslinked portion is 1:99-99:1, the porosity of the non-crosslinked portion is greater than the porosity of the crosslinked portion, and the porosity difference is greater than or equal to 5%; the method for preparing the biological patch of this application may further include the following step: mixing an aqueous solution of a polysaccharide polymer compound and a crosslinking agent... The mixture is combined to obtain a mixed solution; the mixed solution is placed on any surface of the matrix material, allowing the mixed solution to gradually penetrate into the matrix material to a preset depth for a gradient cross-linking reaction to form a cross-linked part and a non-cross-linked part A. After cleaning, a prefabricated biological patch is obtained; an adhesive is placed on the surface of the non-cross-linked part A of the prefabricated biological patch, and then a new matrix material is bonded to the prefabricated biological patch, so that the non-cross-linked part A and the new matrix material together constitute the non-cross-linked part. After water bath and freeze-drying, a biological patch is obtained, wherein the thickness ratio of the non-cross-linked part to the cross-linked part is 1:10-10:1, the porosity of the non-cross-linked part is greater than the porosity of the cross-linked part, and the porosity difference is greater than or equal to 5%.

[0021] In one embodiment, the method for preparing the biological patch further satisfies at least one of the following conditions: (1) the mass fraction of the polysaccharide polymer in the aqueous solution is 3%-10%, wherein the aqueous solution of the polysaccharide polymer is selected from at least one of chitosan aqueous solution, chitosan derivative aqueous solution, cellulose aqueous solution, cellulose derivative aqueous solution, guar gum aqueous solution, tamarind gum aqueous solution, sodium carboxymethyl cellulose aqueous solution, and sodium carboxymethyl starch aqueous solution; (2) the viscosity of the mixed solution is 0.01 Pa·s-40000 Pa·s, and the pH value is 8-14; (3) the mixed solution contains... The mass fraction of the crosslinking agent is 0.1%-5%, wherein the crosslinking agent is selected from at least one of aldehyde crosslinking agents, carbomer, and glycidyl ether; (4) the mass fraction of the polysaccharide polymer in the mixed solution is 0.1%-5%; (5) the gradient crosslinking reaction temperature is 25℃-50℃, and the gradient crosslinking reaction time is 0.5h-48h; (6) the cleaning treatment steps include: first washing with water, then alcohol replacement, then soaking in an aqueous solution of anionic descaling agent with a mass fraction of 0.2%-1% for swelling for 1h-2h, and finally placing it in water; (7) the matrix material is selected from decellularized dermal matrix or collagen.

[0022] This application also discloses a method for preparing a biological patch, comprising the following steps: mixing an aqueous solution of a polysaccharide polymer compound and a crosslinking agent, then adding an alkaline solution to adjust to alkalinity, thereby obtaining a mixed solution; placing the mixed solution on either surface of a first matrix material and a second matrix material, allowing the mixed solution to gradually penetrate into the first matrix material and the second matrix material to a predetermined depth to perform a gradient crosslinking reaction, forming a first gradient crosslinked portion, a first non-crosslinked portion, a second gradient crosslinked portion, and a second non-crosslinked portion; then washing and freeze-drying to obtain a first intermediate and a second intermediate; and placing an adhesive on the first gradient crosslinked portion of the first intermediate. The first intermediate and / or the second intermediate are separated and / or bonded together, such that the first and second gradient cross-linked portions together constitute a cross-linked portion, and the first and second non-cross-linked portions together constitute a non-cross-linked portion, with the cross-linked portion located between the first and second non-cross-linked portions. After water bath and freeze-drying, a biological patch is obtained, wherein the thickness ratio of the non-cross-linked portion to the cross-linked portion is 1:99-99:1, the porosity of the non-cross-linked portion is greater than the porosity of the cross-linked portion, and the porosity difference is greater than or equal to 5%.

[0023] In one embodiment, the method for preparing the biological patch further satisfies at least one of the following conditions: (1) the mass fraction of the polysaccharide polymer in the aqueous solution is 3%-10%, wherein the aqueous solution of the polysaccharide polymer is selected from at least one of chitosan aqueous solution, chitosan derivative aqueous solution, cellulose aqueous solution, cellulose derivative aqueous solution, guar gum aqueous solution, tamarind gum aqueous solution, sodium carboxymethyl cellulose aqueous solution, and sodium carboxymethyl starch aqueous solution; (2) the viscosity of the mixed solution is 0.01 Pa·s-40000 Pa·s, and the pH value is 8-14; (3) the mass fraction of the crosslinking agent in the mixed solution is 3%-10%. The mass fraction is 0.1%-5%, wherein the crosslinking agent is selected from at least one of aldehyde crosslinking agent, carboimide, and glycidyl ether; (4) the mass fraction of the polysaccharide polymer compound in the mixed solution is 0.1%-5%; (5) the gradient crosslinking reaction temperature is 25℃-50℃, and the gradient crosslinking reaction time is 0.5h-48h; (6) the cleaning treatment steps include: first washing with water, then alcohol replacement, then soaking in an anionic descaling agent aqueous solution with a mass fraction of 0.2%-1% for swelling for 1h-2h, and finally placing it in water; (7) the first matrix material and the second matrix material are independently selected from dermal decellularized matrix or collagen.

[0024] This application also discloses a method for preparing a biological patch, comprising the following steps: S1, mixing an aqueous solution of a polysaccharide polymer compound and a crosslinking agent to obtain a mixed solution; S2, placing the mixed solution on any surface of a matrix material, allowing the mixed solution to gradually penetrate into the matrix material to a preset depth for a gradient crosslinking reaction, followed by washing and freeze-drying to obtain a biological patch, wherein the biological patch comprises a crosslinked portion and a non-crosslinked portion, the thickness ratio of the non-crosslinked portion to the crosslinked portion of the biological patch is 1:99-99:1, the porosity of the non-crosslinked portion of the biological patch is greater than the porosity of the crosslinked portion of the biological patch, and the porosity difference is greater than or equal to 5%.

[0025] In one embodiment, step S2 includes: S21, placing the mixed solution on any surface of the matrix material, allowing the mixed solution to gradually penetrate into the matrix material to a preset depth to perform a gradient crosslinking reaction to form a crosslinked portion and a non-crosslinked portion, and then cleaning to obtain a prefabricated bio-patch; S22, placing an adhesive on the surface of the non-crosslinked portion of the prefabricated bio-patch, bonding the new matrix material to the non-crosslinked portion of the prefabricated bio-patch, and then performing a water bath and freeze-drying treatment to obtain a bio-patch, wherein the bio-patch includes a crosslinked portion and a non-crosslinked portion, the thickness ratio of the non-crosslinked portion to the crosslinked portion of the bio-patch is 1:10-10:1, and the porosity of the non-crosslinked portion of the bio-patch is greater than the porosity of the crosslinked portion of the bio-patch.

[0026] In one embodiment, the matrix material in S2 includes a first matrix material and a second matrix material. S2 includes the following steps: S21', placing the mixed solution on either surface of the first matrix material and the second matrix material, allowing the mixed solution to gradually penetrate into the first matrix material and the second matrix material to a preset depth for a gradient crosslinking reaction, followed by washing and freeze-drying to obtain a first intermediate and a second intermediate, wherein the first intermediate includes a first gradient crosslinked portion and a first non-crosslinked portion, and the second intermediate includes a second gradient crosslinked portion and a second non-crosslinked portion; S22', placing an adhesive on the first gradient crosslinked portion and / or The second gradient cross-linked portion of the second intermediate is applied to the surface of the second intermediate. Then, the first intermediate and the second intermediate are bonded together and subjected to water bath and freeze-drying to obtain a biological patch. The biological patch includes a cross-linked portion and a non-cross-linked portion. The cross-linked portion of the biological patch includes the first gradient cross-linked portion and the second gradient cross-linked portion. The non-cross-linked portion of the biological patch includes the first non-cross-linked portion and the second non-cross-linked portion, and the cross-linked portion of the biological patch is located between the first non-cross-linked portion and the second non-cross-linked portion. The thickness ratio of the non-cross-linked portion to the cross-linked portion of the biological patch is 1:10-10:1.

[0027] In one embodiment, the method for preparing the biological patch further satisfies at least one of the following conditions: (1) the mass fraction of the polysaccharide polymer in the aqueous solution is 3%-10%, wherein the aqueous solution of the polysaccharide polymer is selected from at least one of chitosan aqueous solution, chitosan derivative aqueous solution, cellulose aqueous solution, cellulose derivative aqueous solution, guar gum aqueous solution, tamarind gum aqueous solution, sodium carboxymethyl cellulose aqueous solution, and sodium carboxymethyl starch aqueous solution; (2) the viscosity of the mixed solution is 0.01 Pa·s-40000 Pa·s, and the pH value is 8-14; (3) the mixed solution contains... The mass fraction of the crosslinking agent is 0.1%-5%, wherein the crosslinking agent is selected from at least one of aldehyde crosslinking agents, carbomer, and glycidyl ether; (4) the mass fraction of the polysaccharide polymer in the mixed solution is 0.1%-5%; (5) the gradient crosslinking reaction temperature is 25℃-50℃, and the gradient crosslinking reaction time is 0.5h-48h; (6) the cleaning treatment steps include: first washing with water, then alcohol replacement, then soaking in an aqueous solution of anionic descaling agent with a mass fraction of 0.2%-1% for swelling for 1h-2h, and finally placing it in water; (7) the matrix material is selected from decellularized dermal matrix or collagen.

[0028] This application also discloses the application of a biological patch as described above in the repair of tissue damage.

[0029] The biological patch of this application, by setting non-crosslinked and crosslinked portions and limiting the relationship between the thickness of the non-crosslinked and crosslinked portions and their porosity, can effectively regulate the overall degradation rate of the biological patch in vivo by utilizing the differences in degradation time and collagen bioactivity between the non-crosslinked and crosslinked portions, and control it within a suitable range. This balances the relationship between collagen bioactivity and degradation time in the biological patch, enabling the biological patch to prolong its degradation time in vivo, maintain good mechanical properties while retaining high collagen bioactivity, and match the degradation rate in vivo with the tissue repair process, thereby better promoting the comprehensive repair of injured tissue.

[0030] Details of one or more embodiments of this application are set forth in the following drawings and description. Other features, objects, and advantages of this application will become apparent from the specification, drawings, and claims. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 is a schematic diagram of the structure of the biological patch according to the first embodiment of this application.

[0033] Figure 2 is a schematic diagram of the structure of the biological patch according to the second embodiment of this application.

[0034] Figure 3 is a schematic diagram of the pore distribution in the cross-linked part of Figure 2.

[0035] Figure 4 is a schematic diagram of the structure of the biological patch according to the third embodiment of this application.

[0036] Figure 5 is a schematic diagram of the cross-linked portion in Figure 4.

[0037] Figure 6 is a schematic diagram of the pore distribution in the cross-linked portion of Figure 5.

[0038] Figure 7 is an electron microscope image of the longitudinal cross-section of the biological patch prepared in Example 1 of this application.

[0039] Reference numerals: 1. Non-crosslinked portion; 2. Crosslinked portion; 3. First non-crosslinked portion; 4. Second non-crosslinked portion. Detailed Implementation

[0040] To facilitate understanding of this application, it will be described in more detail below. However, it should be understood that this application can be implemented in many different forms and is not limited to the embodiments or examples described herein. Rather, these embodiments or examples are provided to provide a more thorough and complete understanding of the disclosure of this application.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular implementations or embodiments only and is not intended to be limiting of this application. The optional range of the term "and / or" as used herein includes any one of two or more of the related listed items, as well as any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all related listed items.

[0042] As shown in Figures 1-6, the biological patch provided in this application, along the thickness direction of the biological patch (i.e., the direction indicated by the arrow in Figure 1), includes a non-crosslinked portion 1 and a crosslinked portion 2. The non-crosslinked portion 1 is used for direct contact with the tissue to be repaired. It can be understood that when the biological patch is implanted in the body for the repair of damaged tissue, the non-crosslinked portion 1, due to its high collagen bioactivity and faster degradation performance, directly contacts the damaged tissue after implantation, rapidly releasing cytokines required in the early stages of tissue repair and inducing rapid tissue ingrowth in the early post-implantation period. Meanwhile, the crosslinked portion has relatively low collagen bioactivity and a longer degradation time, which prolongs the overall degradation time of the biological patch in the body, facilitating comprehensive repair of damaged tissue.

[0043] Wherein, the thickness ratio of the non-crosslinked portion 1 to the crosslinked portion 2 is 1:99-99:1, optionally, the thickness ratio is 1:50-50:1, the porosity of the non-crosslinked portion 1 is greater than the porosity of the crosslinked portion 2, and the porosity difference is greater than or equal to 5%, optionally, the porosity difference is greater than or equal to 10%, and further optionally, the porosity difference is greater than or equal to 20%.

[0044] In this application, by limiting the thickness of the non-crosslinked portion 1 and the crosslinked portion 2 and the relationship between their porosities, the differences in degradation time and collagen bioactivity between the non-crosslinked portion 1 and the crosslinked portion 2 can be effectively utilized to regulate the overall degradation rate of the bio-patch in vivo and control it within a suitable range. This balances the relationship between collagen bioactivity and degradation time in the bio-patch, enabling the bio-patch to prolong its degradation time in vivo, maintain good mechanical properties while retaining high collagen bioactivity, and match its degradation rate in vivo with the tissue repair process, thereby better promoting the comprehensive repair of injured tissues.

[0045] Optionally, the average porosity of the non-crosslinked portion 1 is 40%-90%, specifically, the average porosity of the non-crosslinked portion 1 includes, but is not limited to, 40%, 50%, 60%, 70%, 80%, and 90%.

[0046] Furthermore, the average porosity of the cross-linked portion 2 is 0.1%-40%, specifically, the average porosity of the cross-linked portion 2 includes, but is not limited to, 1%, 5%, 10%, 20%, 30%, and 40%. This configuration allows the bio-patch to better combine high collagen bioactivity and mechanical properties by adjusting the average porosity of the non-cross-linked portion 1 and the average porosity of the cross-linked portion 2.

[0047] It is understood that in this application, the porosity of the cross-linked portion 2 will affect the degradation rate of the biological patch during tissue repair and the level of collagen bioactivity. In this application, the porosity of the cross-linked portion 2 can be one of the following two cases in the direction from the cross-linked portion 2 to the non-cross-linked portion 1.

[0048] In some embodiments, the porosity of the cross-linked portion 2 is the same from the direction of the non-cross-linked portion 1, as shown in Figure 1. Further, the thickness ratio of the non-cross-linked portion 1 to the cross-linked portion 2 is 1:9-9:1. This configuration facilitates better control of the overall degradation time and collagen bioactivity of the biological patch, and better promotes its degradation rate in vivo to match the tissue repair process.

[0049] In some embodiments, the porosity of the cross-linked portion 2 increases in a gradient from the cross-linked portion 2 to the non-cross-linked portion 1, as shown in Figures 2 and 3. With this configuration, the cross-linked portion 2 can be gradient-crosslinked in the thickness direction by controlling the change in porosity. This results in a greater distance from the non-cross-linked portion 1 and a longer degradation time, thereby guiding the gradient degradation of the biological patch in the thickness direction. This gradient degradation can better match the tissue ingrowth process, thus providing better mechanical support throughout the tissue regeneration process, while also degrading appropriately to provide space for tissue ingrowth, thereby achieving better comprehensive repair of injured tissue.

[0050] Optionally, when the porosity of the cross-linked portion 2 increases in a gradient, the thickness ratio of the non-cross-linked portion 1 to the cross-linked portion 2 is 1:99-99:1, optionally, the thickness ratio is 1:10-10:1, and further optionally, the thickness ratio is 1:4-4:1. This configuration helps to further prolong the degradation time of the biological patch in vivo, improve mechanical properties, and better maintain high collagen bioactivity, thereby better repairing injured tissue.

[0051] Optionally, when the porosity increases in a gradient, the cross-linked portion 2 contains a polysaccharide polymer, and the average degree of cross-linking of the polysaccharide polymer is 0.1%-50%. This setting is beneficial for further controlling the porosity of the cross-linked portion.

[0052] Furthermore, the polysaccharide polymer is selected from biodegradable polysaccharide polymers, specifically from at least one of chitosan, chitosan derivatives, cellulose, cellulose derivatives, guar gum, tamarind gum, sodium carboxymethyl cellulose, and sodium carboxymethyl starch. This configuration extends the degradation time of the bio-patch in vivo, providing sustained mechanical properties while maintaining good biocompatibility with the human body, thus improving the biosafety of the bio-patch.

[0053] Figures 4-6 illustrate the structure of the biological patch according to the third embodiment of this application. Referring to Figures 4 and 6, the non-crosslinked portion 1 includes a first non-crosslinked portion 3 and a second non-crosslinked portion 4. The crosslinked portion 2 is located between the first non-crosslinked portion 3 and the second non-crosslinked portion 4. From the first non-crosslinked portion 3 towards the second non-crosslinked portion 4, the porosity of the crosslinked portion 2 decreases first and then increases. This arrangement ensures that both sides of the biological patch are non-crosslinked portions 1 with high collagen bioactivity and faster degradation performance. When implanted into the body, the two sides of the biological patch directly contact the damaged tissue, inducing rapid tissue ingrowth from both sides in the early post-implantation period. The degradation time of the middle crosslinked portion is longer, and the greater the distance from the first and second non-crosslinked portions, the longer the degradation time. Therefore, it can further guide tissue ingrowth towards the center of the biological patch in the thickness direction, ultimately achieving tissue healing on both sides.

[0054] In this application, the thickness ratio of the first non-crosslinked portion 3 to the second non-crosslinked portion 4 is 1:99-99:1. Specifically, the thickness ratio of the first non-crosslinked portion 3 to the second non-crosslinked portion 4 includes, but is not limited to, 1:50, 1:9, 1:5, 1:1, 5:1, 9:1, and 50:1, and can be selected as 1:50-50:1, further selected as 1:9-9:1, and even further selected as 1:5-5:1. This setting is beneficial for better promoting the rapid healing and regeneration of tissues on both sides in the body.

[0055] It is understood that the thicknesses of the first non-crosslinked portion 3 and the second non-crosslinked portion 4 in this application can be the same or different. This arrangement helps to improve the consistency of uniform tissue growth on both sides.

[0056] In one embodiment, the non-crosslinked portion 1 and the crosslinked portion 2 are integrally formed. This arrangement simplifies the structure.

[0057] Optionally, an adhesive layer is further provided between the non-crosslinked portion 1 and the crosslinked portion 2. This arrangement helps to improve the bonding force between the non-crosslinked portion 1 and the crosslinked portion 2.

[0058] The thickness of the adhesive layer is 0.1%-20% of the total thickness of the substrate. Specifically, the thickness of the adhesive layer includes, but is not limited to, 0.1%, 0.5%, 1%, 5%, 10%, 15%, and 20%.

[0059] Optionally, the material of the matrix is ​​selected from decellularized dermal matrix or collagen, and may be selected from decellularized dermal matrix.

[0060] Optionally, the thickness of the biological patch is 0.5mm-5.5mm; specifically, the thickness of the biological patch includes, but is not limited to, 0.5mm, 1mm, 1.5mm, 2mm, 2.5mm, 3mm, 3.5mm, 4mm, 4.5mm, 5mm, and 5.5mm. This design effectively ensures the mechanical properties of the biological patch while improving its adhesion to the damaged tissue.

[0061] Optionally, the average pore size of the biological patch is 0.1μm-500μm, and can be selected as 5μm-200μm; specifically, the average pore size of the biological patch includes, but is not limited to, 5μm, 10μm, 20μm, 30μm, 40μm, 50μm, 60μm, 100μm, 120μm, 150μm, and 200μm.

[0062] Furthermore, the average porosity of the biological patch is 10%-80%; specifically, the average porosity of the biological patch includes, but is not limited to, 10%, 20%, 30%, 40%, 50%, 60%, 70%, and 80%.

[0063] In this application, by setting appropriate thickness, pore size, and average porosity, the biological patch not only better simulates the structure of normal tissue, providing scaffolding and other cell matrix components for cell growth, and better supporting cell populations conducive to tissue repair and regeneration, thus promoting tissue repair and regeneration; but also possesses excellent suture strength, tensile strength, and tear resistance, providing the mechanical reinforcement required for tissue regeneration and repair. Specifically, the biological patch has a suture strength of 8N-80N, a maximum tensile force of 10N-180N, and a tear strength of 5N-50N.

[0064] In addition, this application also provides a method for preparing the biological patch with the structure shown in Figure 1, which specifically includes the following steps.

[0065] S11, the matrix material is placed in an aqueous solution of a crosslinking agent and subjected to a crosslinking reaction at a temperature of 25℃-35℃ for 10h-15h to obtain a crosslinked matrix material, wherein the mass fraction of the crosslinking agent in the aqueous solution of the crosslinking agent is 0.1%-2%; it can be understood that in this process, the crosslinking agent reacts with the collagen molecules in the matrix material to form a crosslinked portion 2.

[0066] In one embodiment, the matrix material is selected from decellularized dermal matrix or collagen.

[0067] Further, the crosslinking agent is selected from at least one of aldehyde crosslinking agents, carboimide, genipin, glycidyl ether, or epoxy crosslinking agents, and may be selected from aldehyde crosslinking agents, carboimide, or glycidyl ether, and may be further selected from glutaraldehyde, carboimide, or 1,4-butanediol glycidyl ether.

[0068] It should be noted that different types of crosslinking agents require different environments to crosslink with the collagen active substances in the matrix material. Therefore, the acidity or alkalinity of the crosslinking agent aqueous solution can be adjusted according to the type of crosslinking agent. For example, when the crosslinking agent is selected from aldehyde crosslinking agents or glycidyl ethers, an alkaline solution can be added to the crosslinking agent aqueous solution to adjust the pH value to 12-13; when the crosslinking agent is selected from carboimide, the crosslinking agent aqueous solution can be controlled to be weakly acidic.

[0069] S12, the cross-linked matrix material is washed with water until neutral, and then freeze-dried to obtain the freeze-dried matrix material.

[0070] S13, the adhesive is placed on the surface of the new matrix material, and then the freeze-dried matrix material is placed on the surface of the matrix material containing the adhesive. After freeze-drying, a cross-linked portion 2 and a non-cross-linked portion 1 are formed, resulting in the biological patch shown in Figure 1. The thickness ratio of the non-cross-linked portion 1 to the cross-linked portion 2 is 1:9-9:1. The porosity of the non-cross-linked portion is greater than that of the cross-linked portion, and the porosity difference is greater than or equal to 10%. It can be understood that the cross-linked matrix material serves as the cross-linked portion 2, and the cross-linking of this cross-linked portion 2 is uniformly distributed, i.e., the porosity is the same. The new matrix material serves as the non-cross-linked portion 1.

[0071] Furthermore, the adhesive is selected from fibrin glue or collagen solution. This configuration not only improves the bonding force between the non-crosslinked portion 1 and the crosslinked portion 2, but also provides the biological patch with nutrients for cell growth.

[0072] This application also provides a method for preparing the biological patch with the structure shown in Figure 2. The biological patch with this structure can be prepared by referring to either of the following two preparation methods, as detailed below.

[0073] The first preparation method includes the following steps: mixing an aqueous solution of a polysaccharide polymer compound and a crosslinking agent to obtain a mixed solution; placing the mixed solution on any surface of a matrix material, allowing the mixed solution to gradually penetrate into the matrix material to a preset depth to carry out a gradient crosslinking reaction to form a crosslinked part 2 and a non-crosslinked part 1; after cleaning and freeze-drying, a biological patch is obtained, wherein the thickness ratio of the non-crosslinked part 1 to the crosslinked part 2 is 1:99-99:1, the porosity of the non-crosslinked part is greater than the porosity of the crosslinked part, and the porosity difference is greater than or equal to 5%.

[0074] The second preparation method includes the following steps: mixing an aqueous solution of a polysaccharide polymer compound and a crosslinking agent to obtain a mixed solution; placing the mixed solution on any surface of a matrix material, allowing the mixed solution to gradually penetrate into the matrix material to a preset depth for a gradient crosslinking reaction to form a crosslinked portion 2 and a non-crosslinked portion A; cleaning to obtain a prefabricated biological patch; placing an adhesive on the surface of the non-crosslinked portion A of the prefabricated biological patch, and then bonding a new matrix material to the prefabricated biological patch, so that the non-crosslinked portion A and the new matrix material together constitute the non-crosslinked portion 1; and obtaining a biological patch by water bath and freeze-drying, wherein the thickness ratio of the non-crosslinked portion 1 to the crosslinked portion 2 is 1:10-10:1, the porosity of the non-crosslinked portion is greater than the porosity of the crosslinked portion, and the porosity difference is greater than or equal to 5%.

[0075] Optionally, the matrix material is selected from decellularized dermal matrix or collagen.

[0076] It is understood that, in this application, the two preparation methods described above can be selected to prepare the biological patch with the structure shown in Figure 2, depending on actual needs.

[0077] Furthermore, this application also provides a method for preparing the biological patch with the structure shown in Figure 4, comprising the following steps: mixing an aqueous solution of a polysaccharide polymer compound and a crosslinking agent, then adding an alkaline solution to adjust to alkalinity to obtain a mixed solution; placing the mixed solution on either surface of a first matrix material and a second matrix material, allowing the mixed solution to gradually penetrate into the first matrix material and the second matrix material to a preset depth for a gradient crosslinking reaction to form a first gradient crosslinked portion, a first non-crosslinked portion 3, a second gradient crosslinked portion, and a second non-crosslinked portion 4; then washing and freeze-drying to obtain a first intermediate and a second intermediate; placing an adhesive on the first gradient crosslinked portion and / or the first intermediate. Alternatively, the second gradient cross-linked portion of the second intermediate is applied to the surface of the first intermediate and the second intermediate, and then the first intermediate and the second intermediate are bonded together, such that the first gradient cross-linked portion and the second gradient cross-linked portion together constitute the cross-linked portion 2, and the first non-cross-linked portion 3 and the second non-cross-linked portion 4 together constitute the non-cross-linked portion 1, and the cross-linked portion 2 is located between the first non-cross-linked portion 3 and the second non-cross-linked portion 4. After water bath and freeze drying, a biological patch as shown in Figure 4 is obtained, wherein the thickness ratio of the non-cross-linked portion 1 to the cross-linked portion 2 is 1:99-99:1, the porosity of the non-cross-linked portion is greater than the porosity of the cross-linked portion, and the porosity difference is greater than or equal to 5%.

[0078] It should be noted that the first matrix material and the second matrix material used in the above method can be obtained by cutting a matrix material into two parts, or they can be two complete matrix materials. The first matrix material and the second matrix material are independently selected from decellularized dermal matrix or collagen.

[0079] It is understood that in this application, the preparation methods of both the structure shown in Figure 2 and the structure shown in Figure 4 utilize the gravity and fluidity of the mixed solution to gradually penetrate from one side of the matrix material inwards. This causes the cross-linking agent in the mixed solution to achieve a gradient distribution along the thickness direction of the matrix material under the action of polysaccharide polymers. Consequently, the reaction degree between collagen and the cross-linking agent along the thickness direction of the matrix material gradually weakens, resulting in different degrees of cross-linking and obtaining a biological patch with a gradient distribution of porosity.

[0080] It should be noted that in this application, the gradient crosslinking reduces the porosity of the matrix material mainly because the crosslinking agent acts as a bridge between collagen molecules, causing previously distant collagen molecules to form a denser mesh structure under the overlap of the crosslinking agent, thereby reducing the porosity of the biological patch. On the other hand, polysaccharide polymers contribute very little to the dense collagen mesh. Their main function is to increase the viscosity of the crosslinking agent solution so that the crosslinking agent can slowly penetrate from one surface of the matrix material into the interior of the matrix material, causing the crosslinking dosage to show a gradient distribution from more to less or from less to more along the thickness direction of the matrix material.

[0081] Optionally, the mass fraction of the polysaccharide polymer in the aqueous solution is 3%-10%, which is beneficial for achieving better gradient crosslinking.

[0082] Furthermore, the aqueous solution of the polysaccharide polymer is selected from at least one of chitosan aqueous solution, chitosan derivative aqueous solution, cellulose aqueous solution, cellulose derivative aqueous solution, guar gum aqueous solution, tamarind gum aqueous solution, sodium carboxymethyl cellulose aqueous solution, and sodium carboxymethyl starch aqueous solution.

[0083] Optionally, the viscosity of the mixed solution is 0.01 Pa·s-40000 Pa·s. This setting is beneficial for controlling the penetration crosslinking time and degree of crosslinking, and controlling the gradient distribution of the porosity of the crosslinked part 2.

[0084] Optionally, the pH of the mixed solution is 8-14. This setting is beneficial for promoting the cross-linking reaction.

[0085] Optionally, the mass fraction of the crosslinking agent in the mixed solution is 0.1%-5%; the mass fraction of the polysaccharide polymer compound in the mixed solution is 0.1%-5%. With this setting, the gradient crosslinking reaction between the crosslinking agent and the collagen active substances in the matrix material can be controlled by controlling the amount of crosslinking agent and the polysaccharide polymer compound, thereby forming crosslinked parts 2 with different crosslinking characteristics.

[0086] Furthermore, the crosslinking agent is selected from at least one of aldehyde crosslinking agents, carboimide, and glycidyl ether.

[0087] It should be noted that, depending on the type of crosslinking agent and the different environments required for crosslinking with the collagen active substances in the matrix material, the acidity or alkalinity of the mixed solution can be adjusted according to the type of crosslinking agent. For example, when the crosslinking agent is selected from aldehyde crosslinking agents or glycidyl ethers, an alkaline solution can be added to the mixed solution to adjust the pH value to 12-13; when the crosslinking agent is selected from carboimide, the mixed solution can be controlled to be weakly acidic.

[0088] Optionally, the cleaning process includes: first, water washing; then, alcohol replacement; followed by soaking in a 0.2%-1% (w / w) anionic descaling agent aqueous solution for swelling for 1-2 hours; and finally, immersion in water, wherein the anionic descaling agent aqueous solution is selected from sodium dodecyl sulfate solution. This setup effectively removes unreacted crosslinking agents and polysaccharide polymers from the substrate material.

[0089] Optionally, the gradient crosslinking reaction temperature is 25℃-50℃, and the gradient crosslinking reaction time is 0.5h-48h. This setting allows for the control of the thickness of the crosslinked portion 2 and the non-crosslinked portion 1.

[0090] It should be noted that the matrix materials used in this application are all freeze-dried matrix materials, and the preparation of such matrix materials is a conventional technical method, so it will not be described in detail here.

[0091] In addition, this application also provides the application of the biological patch as described above in the repair of tissue damage.

[0092] Specifically, in one embodiment, this application also provides the application of a biological patch in tendon soft tissue repair.

[0093] The following specific embodiments will further illustrate the biological patch, its preparation method, and its application. However, those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Unless otherwise specified, specific conditions in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used without specified manufacturers are all commercially available conventional products.

[0094] It should also be noted that the average porosity of the non-crosslinked portion, the average porosity of the crosslinked portion, and the average porosity of the biological patch involved in the embodiments and comparative examples in this application were all measured using an electron scanning microscope (ZEISS Sigma 300, Germany).

[0095] Example 1

[0096] A 5% (w / w) aqueous solution of hydroxyethyl cellulose and glutaraldehyde were mixed to obtain a first mixture, in which the mass fraction of hydroxyethyl cellulose was 2% and the mass fraction of glutaraldehyde was 2%. Then, 5 mol / L sodium hydroxide was added to adjust the pH of the mixture to 12 and stirred until homogeneous to obtain a second mixture.

[0097] The second mixed solution was applied to the surface of a dry dermal decellularized matrix, and then a cross-linking reaction was carried out at 40°C for 6 hours to form the cross-linked part and the non-cross-linked part A, thus obtaining the cross-linked dermal decellularized matrix.

[0098] The cross-linked decellularized dermal matrix was washed with water, and after being repeatedly replaced twice with ethanol, it was soaked in a 0.5% sodium dodecyl sulfate solution to swell for 1.5 hours, and then replaced with purified water to obtain a pre-made biological patch.

[0099] A collagen solution was placed on the surface of the non-crosslinked portion A of the prefabricated biological patch. Then, a new decellularized dry dermal matrix was bonded to the prefabricated biological patch, so that the non-crosslinked portion A and the new decellularized dry dermal matrix together constitute the non-crosslinked portion. After being in a water bath at 40°C for 5 hours, the patch was freeze-dried to obtain the biological patch. The thickness ratio of the non-crosslinked portion to the crosslinked portion was 11:9. The average porosity of the non-crosslinked portion was 81.15%, and the average porosity of the crosslinked portion was 0.5%-15%. The thickness of the biological patch was 3 mm, the average pore size was 55.71 μm, and the average porosity was 49.13%.

[0100] The longitudinal section of the biological patch prepared in this embodiment was examined using a scanning electron microscope. The results are shown in Figure 7. As can be seen from Figure 7, the porosity of the non-crosslinked part is greater than that of the crosslinked part, and the porosity difference is greater than 40%.

[0101] Example 2

[0102] An 8% (w / w) aqueous solution of hydroxyethyl cellulose and 1,4-butanediol diglycidyl ether were mixed to obtain a first mixture, in which the mass fraction of hydroxyethyl cellulose was 4% and the mass fraction of 1,4-butanediol diglycidyl ether was 4%. Then, 5 mol / L sodium hydroxide was added to adjust the pH of the solution to 10 and stirred until homogeneous to obtain a second mixture.

[0103] The second mixed solution was applied to the surface of the dried decellularized dermal matrix, and then a cross-linking reaction was carried out at 35°C for 8 hours to form a cross-linked part and a non-cross-linked part, thus obtaining the cross-linked decellularized dermal matrix.

[0104] The cross-linked decellularized dermal matrix was washed with water, repeatedly replaced twice with ethanol, and then soaked in a 0.8% sodium dodecyl sulfate solution for swelling for 1.5 hours. After being replaced with purified water, it was lyophilized to obtain a biological patch. The thickness ratio of the non-cross-linked to the cross-linked portion was 1:19. The average porosity of the non-cross-linked portion was 72.42%, and the average porosity of the cross-linked portion was 0.1%-10%. The biological patch had a thickness of 1.15 mm, an average pore size of 48.11 μm, and an average porosity of 45.27%.

[0105] Example 3

[0106] The only difference between Example 3 and Example 1 is that a piece of dried decellularized dermal matrix is ​​cut laterally into two parts to obtain a first decellularized dermal matrix and a second decellularized dermal matrix.

[0107] The second mixed solution was placed on either surface of the first and second decellularized dermal matrix, and then cross-linked at 40°C for 4 hours to form a first gradient cross-linked part, a first non-cross-linked part, a second gradient cross-linked part, and a second non-cross-linked part. After washing with water and repeated replacement with ethanol twice, the mixture was soaked in a 0.5% sodium dodecyl sulfate solution for swelling for 1.5 hours, and then replaced with purified water to obtain the first intermediate and the second intermediate.

[0108] A collagen solution was placed on the surface of the first gradient cross-linked portion of the first intermediate and the second gradient cross-linked portion of the second intermediate. The first and second intermediates were then bonded together, such that the first and second gradient cross-linked portions together constitute the cross-linked portion, and the first and second non-cross-linked portions together constitute the non-cross-linked portion. After being in a water bath at 30°C for 6 hours, the mixture was freeze-dried to obtain a biological patch. The thickness ratio of the non-cross-linked portion to the cross-linked portion was 2:1, the thickness ratio of the first non-cross-linked portion to the second non-cross-linked portion was 1:1, the average porosity of the non-cross-linked portion was 74.41%, the average porosity of the cross-linked portion was 2%-20%, the thickness of the biological patch was 3.00 mm, the average pore size was 85.03 μm, and the average porosity was 64.85%.

[0109] Example 4

[0110] The dried decellularized dermal matrix was immersed in a 1% aqueous solution of 1,4-butanediol diglycidyl ether, and then cross-linked at 30°C for 12 hours to form a cross-linked portion, thus obtaining the cross-linked decellularized dermal matrix.

[0111] The cross-linked decellularized dermal matrix was washed with water, and then repeatedly replaced with ethanol twice. After soaking in a 0.5% sodium dodecyl sulfate solution for 1.5 hours to swell, it was replaced with purified water to obtain a pre-made biological patch.

[0112] A collagen solution was placed on the surface of a new decellularized dry dermal matrix. The pre-made biological patch and the new decellularized dry dermal matrix were then bonded together to form a non-crosslinked portion and a crosslinked portion. After incubating in a water bath at 37°C for 5 hours, the patch was freeze-dried to obtain the biological patch. The thickness ratio of the non-crosslinked portion to the crosslinked portion was 1:1. The average porosity of the non-crosslinked portion was 76.14%, and the average porosity of the crosslinked portion was 16.84%. The biological patch had a thickness of 3.00 mm, an average pore size of 64.2 μm, and an average porosity of 58.88%.

[0113] Example 5

[0114] A 5% (w / w) aqueous solution of hydroxyethyl cellulose and 1,4-butanediol diglycidyl ether were mixed to obtain a first mixture, in which the mass fraction of hydroxyethyl cellulose was 3% and the mass fraction of 1,4-butanediol diglycidyl ether was 3%. Then, 5 mol / L sodium hydroxide was added to adjust the pH of the solution to 10 and stirred until homogeneous to obtain a second mixture.

[0115] The second mixed solution was applied to the surface of a dry collagen patch, and then a cross-linking reaction was carried out at 30°C for 12 hours to form a cross-linked portion and a non-cross-linked distribution, thus obtaining a cross-linked collagen patch.

[0116] The cross-linked collagen patch was washed with water, repeatedly replaced twice with ethanol, and then soaked in a 0.8% sodium dodecyl sulfate solution for swelling for 1.5 hours. After being replaced with purified water, it was freeze-dried to obtain the biological patch. The thickness ratio of the non-cross-linked portion to the cross-linked portion was 3:10. The average porosity of the non-cross-linked portion was 74.31%, and the average porosity of the cross-linked portion was 35%-55%. The thickness of the biological patch was 1.15 mm, the average pore size was 120.24 μm, and the average porosity was 67.55%.

[0117] Comparative Example 1

[0118] Compared with Example 1, Comparative Example 1 differs only in that the second mixed solution is applied to the surface of a dry decellularized dermal matrix, and then a cross-linking reaction is carried out at 40°C for 72 hours to form a cross-linked part and a non-cross-linked part A, resulting in a cross-linked decellularized dermal matrix. All other conditions are the same, resulting in a biological patch. The thickness ratio of the non-cross-linked part to the cross-linked part is 0.1:99.9, the average porosity of the non-cross-linked part is 51.41%, the average porosity of the cross-linked part is 0.1%-10%, the thickness of the biological patch is 3.00 mm, the average pore size is 15.23 μm, and the average porosity is 8.95%.

[0119] Comparative Example 2

[0120] Comparative Example 2 differs from Example 1 only in that a 12% (w / w) aqueous solution of hydroxyethyl cellulose and 1,4-butanediol diglycidyl ether are mixed to obtain a first mixture. In the first mixture, the mass fraction of hydroxyethyl cellulose is 11%, and the mass fraction of 1,4-butanediol diglycidyl ether is 1%. Then, 5 mol / L sodium hydroxide is added to adjust the pH of the solution to 12 and the mixture is stirred evenly to obtain a second mixture. The second mixture is applied to the surface of a dry decellularized dermal matrix and then subjected to a cross-linking reaction at 30°C for 0.5 h to form a cross-linked part and a non-cross-linked part A, resulting in a cross-linked decellularized dermal matrix. All other conditions are the same to obtain a biological patch. The thickness ratio of the non-cross-linked part to the cross-linked part is 99.5:0.5. The average porosity of the non-cross-linked part is 87.48%, the average porosity of the cross-linked part is 5%-15%, the thickness of the biological patch is 3.00 mm, the average pore size is 87.15 μm, and the average porosity is 85.94%.

[0121] Comparative Example 3

[0122] A 2% (w / w) aqueous solution of hydroxyethyl cellulose and 1,4-butanediol diglycidyl ether were mixed to obtain a first mixture, in which the mass fraction of hydroxyethyl cellulose was 1.5% and the mass fraction of 1,4-butanediol diglycidyl ether was 0.1%. Then, 5 mol / L sodium hydroxide was added to adjust the pH of the solution to 8 and the mixture was stirred until homogeneous, resulting in a second mixture. The second mixture was applied to the surface of a dried decellularized dermal matrix, and then subjected to a cross-linking reaction at 30°C for 5 hours to form cross-linked and non-cross-linked portions, thus obtaining the cross-linked decellularized dermal matrix. The cross-linked decellularized dermal matrix was washed with water, repeatedly exchanged twice with ethanol, and then soaked in a 0.8% sodium dodecyl sulfate solution for swelling for 1.5 hours. After being replaced with purified water, it was lyophilized to obtain a biological patch. The thickness ratio of the non-cross-linked to the cross-linked portion was 1:4. The average porosity of the non-cross-linked portion was 85.4%, and the average porosity of the cross-linked portion was 81.78%-84.02%. The biological patch had a thickness of 1.15 mm, an average pore size of 98.11 μm, and an average porosity of 84.74%.

[0123] Comparative Example 4

[0124] Comparative Example 4 differs from Example 4 only in that a 0.001% (w / w) solution of 1,4-butanediol diglycidyl ether was applied to the surface of a dried decellularized dermal matrix, followed by a cross-linking reaction at 4°C for 1 hour to form a cross-linked portion, resulting in a cross-linked decellularized dermal matrix. All other conditions were the same, yielding a biological patch. The thickness ratio of the non-cross-linked portion to the cross-linked portion was 1:1. The average porosity of the non-cross-linked portion was 81.30%, the average porosity of the cross-linked portion was 79.03%, the thickness of the biological patch was 3.00 mm, the average pore size was 86.94 μm, and the average porosity was 78.89%.

[0125] The bio-patterns prepared in Examples 1-5 and Comparative Examples 1-4 were subjected to degradation performance tests and mechanical property tests at different degradation time points. The test results are shown in Table 1, and the specific test methods are as follows.

[0126] Degradation performance test:

[0127] The biological patch prepared according to the above embodiments or comparative examples was added to a 1% pancreatic enzyme solution and soaked at 37°C for 24 h and 48 h, then removed, washed, and freeze-dried.

[0128] (1) Test method for suture strength

[0129] Testing instrument: Universal tensile testing machine.

[0130] Cut the sample into 1cm×1cm test samples and use No. 2 polyethylene surgical suture to pass through the edge 3 mm away from the center of the test sample; before testing, soak the test sample in physiological saline for 3 minutes; use the upper pneumatic handle of the universal tensile testing machine to clamp the test sample about 5 mm from the end of the suture needle that has not passed through, and suture it into a semi-loop; clamp the end of the suture on the lower handle, stretch it at 20 mm / min, and record the tensile force-displacement curve; record the maximum value of ce (N) as the suture strength.

[0131] (2) Maximum tensile force test method

[0132] Testing instrument: Universal tensile testing machine.

[0133] Cut the sample into test samples with a length of 5mm and a width of 50mm, and soak the test samples in physiological saline for 5 minutes; clamp the test samples at both ends of the universal testing machine and ensure that the initial distance between the clamps is 3cm; stretch the test samples at a tensile speed of 30mm / min until they break, and record the maximum tensile force ce (N) measured during the test, which is the maximum tensile force.

[0134] (3) Tear strength test method

[0135] Testing instrument: Universal tensile testing machine.

[0136] Prepare a single-sided tear sample with a long side of 70 mm and a short side of 40 mm. Pre-tear the sample 50 mm at the center point of the short side before conducting a tear strength test. The distance between the clamps of the tensile testing machine is 50 mm. Immerse the sample in physiological saline for 3 minutes. Clamp one side of the cut on the sample (approximately 20 mm) with the lower clamp. Fold the sample in half 180° and load the other side of the notch into the upper clamp, ensuring the long side of the sample is parallel to the tensile testing machine. The minimum clamp width is 50 ± 2 mm. Tensile test is performed at a rate of 100 ± 20 mm / min until the sample breaks. Record the force-moment diagram and calculate the tear strength: calculate the average of the peak and trough values ​​of the force, i.e., the tear strength (N).

[0137] Table 1

[0138] It should be noted that the matrix material used in Example 5 is collagen. Compared with the mechanical properties of the decellularized dermal matrix itself, the mechanical properties of collagen are relatively low. Therefore, the suture strength, maximum tensile force and tear strength of the biological patch obtained in Example 5 are necessarily lower than those of the biological patch obtained in the example using the decellularized dermal matrix as the matrix material.

[0139] Furthermore, this application applies the biological patches prepared in Examples 1-5 and Comparative Examples 1-4 to the repair of rotator cuff tissue in New Zealand white rabbits. The specific experimental method is as follows: Rabbits were anesthetized via the marginal ear vein. After successful anesthesia, the skin was prepared at the shoulder joint of the left forelimb. A 4cm incision was made along the midline of the shoulder joint longitudinal axis, and the skin and subcutaneous tissue were dissected layer by layer to expose the supraspinatus tendon. The supraspinatus muscle was removed from the greater tubercle of the humeral head to the muscle belly with a blade, with a range of 1.5cm × 1.5cm. The incision was sutured after direct irrigation with physiological saline. After the rotator cuff defect was created in the experimental group, two 2mm diameter bone marrow tunnels were drilled at the greater tubercle with a spacing of 1.5cm between the two bone marrow tunnels. The cross-linked patches prepared in Examples 1-5 and Comparative Examples 1-4 were sterilized and cut into 1.5cm × 1.5cm sheet samples. One end of the patch was fixed at the greater tubercle by passing through the tunnel using 2-0 tendon sutures, and the other end was sutured to the residual end of the supraspinatus muscle. After rinsing thoroughly with saline solution, the incision was sutured layer by layer. The other right forelimb was left untreated and used as a control.

[0140] Experimental results showed that the biological patches prepared using the embodiments of this application survived for 24 weeks after the repair surgery, with good wound healing and no rejection symptoms such as infection or inflammation. Moreover, from 12 to 24 weeks after the repair surgery, the left shoulder movement of all experimental rabbits was basically normal, and no rupture occurred at the repaired rotator cuff tissue site. In contrast, the experimental rabbits repaired using the biological patches prepared using Comparative Examples 1-4 survived for 24 weeks after the repair surgery, but the wound healing was poor, with cases of aseptic infection such as chronic inflammation or re-tears.

[0141] Therefore, in this application, there is a synergistic relationship between the thickness of the non-crosslinked and crosslinked portions and the difference between the porosity of the non-crosslinked portion and the porosity of the crosslinked portion. Under the synergistic effect of the two, the overall degradation rate of the bio-patch can be effectively controlled within a suitable range, balancing the relationship between collagen bioactivity and degradation time in the bio-patch, so that the bio-patch can prolong its degradation time in vivo, maintain good mechanical properties, and maintain high collagen bioactivity.

[0142] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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.

[0143] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A biological patch, characterized in that, The bio-patch includes a matrix. Along the thickness direction of the bio-patch, the matrix includes a non-crosslinked portion and a crosslinked portion, wherein the thickness ratio of the non-crosslinked portion to the crosslinked portion is 1:99-99:1, the porosity of the non-crosslinked portion is greater than the porosity of the crosslinked portion, and the porosity difference is greater than or equal to 5%.

2. The biological patch of claim 1, wherein, The average porosity of the non-crosslinked portion is 40%-90%; And / or, the average porosity of the cross-linked portion is 0.1%-40%.

3. The biological patch of claim 1, wherein, From the cross-linked portion to the non-cross-linked portion, the porosity of the cross-linked portion is the same, or the porosity of the cross-linked portion increases in a gradient.

4. The biological patch of claim 3, wherein, When the porosity of the cross-linked portions is the same, the thickness ratio of the non-cross-linked portions to the cross-linked portions is 1:9-9:

1.

5. The biological patch of claim 3, wherein, When the porosity of the cross-linked portion increases in a gradient, the thickness ratio of the non-cross-linked portion to the cross-linked portion is 1:99-99:

1.

6. The biological patch of claim 3, wherein, When the porosity increases in a gradient, the cross-linked portion contains a polysaccharide polymer, and the average degree of cross-linking of the polysaccharide polymer is 0.1%-50%.

7. The biological patch of claim 6, wherein, The polysaccharide polymer is selected from at least one of chitosan, chitosan derivatives, cellulose, cellulose derivatives, guar gum, tamarind gum, sodium carboxymethyl cellulose, and sodium carboxymethyl starch.

8. The biological patch of claim 1, wherein, The non-crosslinked portion includes a first non-crosslinked portion and a second non-crosslinked portion. The crosslinked portion is located between the first non-crosslinked portion and the second non-crosslinked portion. From the first non-crosslinked portion to the second non-crosslinked portion, the porosity of the crosslinked portion first decreases and then increases.

9. The biological patch according to claim 8, wherein, The thickness ratio of the first non-crosslinked portion to the second non-crosslinked portion is 1:9-9:

1.

10. The biological patch according to any one of claims 1-9, characterized in that, An adhesive layer is further provided between the non-crosslinked portion and the crosslinked portion, and the thickness of the adhesive layer is 0.1%-20% of the total thickness of the substrate; And / or, the material of the matrix is ​​selected from decellularized dermal matrix or collagen.

11. The biological patch according to claim 10, wherein, The thickness of the biological patch is 0.5mm-5.5mm; And / or, the average pore size of the biological patch is 0.1 μm-500 μm, and the average porosity is 10%-80%.

12. A method for preparing the biological patch according to any one of claims 1-11, characterized in that, Includes the following steps: S1. Mix the aqueous solution of the polysaccharide polymer and the cross-linking agent to obtain a mixed solution; S2. Place the mixed solution on any surface of the matrix material, allowing the mixed solution to gradually penetrate into the matrix material to a predetermined depth for a gradient crosslinking reaction. After cleaning and freeze-drying, a biological patch is obtained. The biological patch includes a crosslinked portion and a non-crosslinked portion. The thickness ratio of the non-crosslinked portion to the crosslinked portion of the biological patch is 1:99-99:1, the porosity of the non-crosslinked portion of the biological patch is greater than the porosity of the crosslinked portion of the biological patch, and the porosity difference is greater than or equal to 5%.

13. The preparation method according to claim 12, wherein, S2 includes: S21. The mixed solution is placed on any surface of the matrix material, and the mixed solution gradually penetrates into the matrix material to a preset depth to carry out a gradient cross-linking reaction. After cleaning, a prefabricated biological patch is obtained. The prefabricated biological patch includes a cross-linked part and a non-cross-linked part of the prefabricated biological patch. S22. Apply adhesive to the surface of the non-crosslinked portion of the prefabricated biological patch, bond the new matrix material to the non-crosslinked portion of the prefabricated biological patch, and then perform water bath and freeze-drying treatment to obtain a biological patch, wherein the biological patch includes the crosslinked portion and the non-crosslinked portion of the biological patch. The thickness ratio of the non-crosslinked portion to the crosslinked portion of the biological patch is 1:10-10:1, and the porosity of the non-crosslinked portion of the biological patch is greater than that of the crosslinked portion.

14. The preparation method according to claim 12, wherein, The matrix material in step S2 includes a first matrix material and a second matrix material, and step S2 includes the following steps: S21' The mixed solution is placed on either surface of the first matrix material and the second matrix material, and the mixed solution is allowed to gradually penetrate into the interior of the first matrix material and the second matrix material to a preset depth to carry out a gradient crosslinking reaction. After washing and freeze-drying, a first intermediate and a second intermediate are obtained, respectively. The first intermediate includes a first gradient crosslinked part and a first non-crosslinked part, and the second intermediate includes a second gradient crosslinked part and a second non-crosslinked part. S22'. An adhesive is placed on the surface of the first gradient crosslinked portion of the first intermediate and / or the second gradient crosslinked portion of the second intermediate, and then the first intermediate and the second intermediate are bonded together. After water bath and freeze-drying, a biological patch is obtained, wherein the biological patch includes a crosslinked portion and a non-crosslinked portion, the crosslinked portion of the biological patch includes the first gradient crosslinked portion and the second gradient crosslinked portion, the non-crosslinked portion of the biological patch includes the first non-crosslinked portion and the second non-crosslinked portion, and the crosslinked portion of the biological patch is located between the first non-crosslinked portion and the second non-crosslinked portion; The thickness ratio of the non-crosslinked portion to the crosslinked portion of the biological patch is 1:10-10:

1.

15. The preparation method according to any one of claims 12-14, wherein, The method for preparing the biological patch also satisfies at least one of the following conditions: (1) The mass fraction of the polysaccharide polymer in the aqueous solution is 3%-10%, wherein the aqueous solution of the polysaccharide polymer is selected from at least one of chitosan aqueous solution, chitosan derivative aqueous solution, cellulose aqueous solution, cellulose derivative aqueous solution, guar gum aqueous solution, tamarind gum aqueous solution, sodium carboxymethyl cellulose aqueous solution, and sodium carboxymethyl starch aqueous solution; (2) The viscosity of the mixed solution is 0.01 Pa·s-40000 Pa·s, and the pH value is 8-14; (3) The mass fraction of the crosslinking agent in the mixed solution is 0.1%-5%, wherein the crosslinking agent is selected from at least one of aldehyde crosslinking agents, carboimide, and glycidyl ether; (4) The mass fraction of the polysaccharide polymer in the mixed solution is 0.1%-5%; (5) The gradient crosslinking reaction temperature is 25℃-50℃, and the gradient crosslinking reaction time is 0.5h-48h; (6) The cleaning process includes: first, water washing; then, alcohol replacement; then, soaking in an aqueous solution of 0.2%-1% anionic descaling agent for 1-2 hours to swell; and finally, placing it in water. (7) The matrix material is selected from decellularized dermal matrix or collagen.

16. A method for preparing the biological patch according to any one of claims 1-11, characterized in that, Includes the following steps: An aqueous solution of a polysaccharide polymer and a cross-linking agent are mixed to obtain a mixed solution; The mixed solution is placed on any surface of the matrix material, allowing the mixed solution to gradually penetrate into the matrix material to a preset depth to carry out a gradient cross-linking reaction, forming a cross-linked part and a non-cross-linked part. After cleaning and freeze-drying, a biological patch is obtained, wherein the thickness ratio of the non-cross-linked part to the cross-linked part is 1:99-99:1, the porosity of the non-cross-linked part is greater than the porosity of the cross-linked part, and the porosity difference is greater than or equal to 5%. Alternatively, an aqueous solution of a polysaccharide polymer and a cross-linking agent can be mixed to obtain a mixed solution; The mixed solution is placed on any surface of the matrix material, allowing the mixed solution to gradually penetrate into the matrix material to a preset depth to carry out a gradient cross-linking reaction to form cross-linked and non-cross-linked parts. After cleaning, a prefabricated biological patch is obtained. An adhesive is placed on the surface of the non-crosslinked portion of the prefabricated biological patch, and then a new matrix material is bonded to the prefabricated biological patch so that the non-crosslinked portion and the new matrix material together constitute the non-crosslinked portion. After water bath and freeze drying, a biological patch is obtained, wherein the thickness ratio of the non-crosslinked portion to the crosslinked portion is 1:10-10:1, the porosity of the non-crosslinked portion is greater than the porosity of the crosslinked portion, and the porosity difference is greater than or equal to 5%.

17. The method for preparing the biological patch according to claim 16, wherein, The method for preparing the biological patch also satisfies at least one of the following conditions: (1) The mass fraction of the polysaccharide polymer in the aqueous solution is 3%-10%, wherein the aqueous solution of the polysaccharide polymer is selected from at least one of chitosan aqueous solution, chitosan derivative aqueous solution, cellulose aqueous solution, cellulose derivative aqueous solution, guar gum aqueous solution, tamarind gum aqueous solution, sodium carboxymethyl cellulose aqueous solution, and sodium carboxymethyl starch aqueous solution; (2) The viscosity of the mixed solution is 0.01 Pa·s-40000 Pa·s, and the pH value is 8-14; (3) The mass fraction of the crosslinking agent in the mixed solution is 0.1%-5%, wherein the crosslinking agent is selected from at least one of aldehyde crosslinking agents, carboimide, and glycidyl ether; (4) The mass fraction of the polysaccharide polymer in the mixed solution is 0.1%-5%; (5) The gradient crosslinking reaction temperature is 25℃-50℃, and the gradient crosslinking reaction time is 0.5h-48h; (6) The cleaning process includes: first, water washing; then, alcohol replacement; then, soaking in an aqueous solution of 0.2%-1% anionic descaling agent for 1-2 hours to swell; and finally, placing it in water. (7) The matrix material is selected from decellularized dermal matrix or collagen.

18. A method for preparing the biological patch according to any one of claims 1-11, characterized in that, Includes the following steps: An aqueous solution of a polysaccharide polymer and a cross-linking agent are mixed to obtain a mixed solution; The mixed solution is placed on either surface of the first matrix material and the second matrix material, and the mixed solution is allowed to gradually penetrate into the interior of the first matrix material and the second matrix material to a preset depth to carry out a gradient cross-linking reaction to form a first gradient cross-linked part, a first non-cross-linked part, a second gradient cross-linked part and a second non-cross-linked part. Then, after washing and freeze-drying, a first intermediate and a second intermediate are obtained. An adhesive is placed on the surface of the first gradient crosslinked portion of the first intermediate and / or the second gradient crosslinked portion of the second intermediate. The first intermediate and the second intermediate are then bonded together, such that the first gradient crosslinked portion and the second gradient crosslinked portion together constitute a crosslinked portion, and the first non-crosslinked portion and the second non-crosslinked portion together constitute a non-crosslinked portion. The crosslinked portion is located between the first non-crosslinked portion and the second non-crosslinked portion. After water bath and freeze-drying, a biological patch is obtained, wherein the thickness ratio of the non-crosslinked portion to the crosslinked portion is 1:10-10:1, the porosity of the non-crosslinked portion is greater than the porosity of the crosslinked portion, and the porosity difference is greater than or equal to 5%.

19. The method for preparing the biological patch according to claim 18, wherein, The method for preparing the biological patch also satisfies at least one of the following conditions: (1) The mass fraction of the polysaccharide polymer in the aqueous solution is 3%-10%, wherein the aqueous solution of the polysaccharide polymer is selected from at least one of chitosan aqueous solution, chitosan derivative aqueous solution, cellulose aqueous solution, cellulose derivative aqueous solution, guar gum aqueous solution, tamarind gum aqueous solution, sodium carboxymethyl cellulose aqueous solution, and sodium carboxymethyl starch aqueous solution; (2) The viscosity of the mixed solution is 0.01 Pa·s-40000 Pa·s, and the pH value is 8-14; (3) The mass fraction of the crosslinking agent in the mixed solution is 0.1%-5%, wherein the crosslinking agent is selected from at least one of aldehyde crosslinking agents, carboimide, and glycidyl ether; (4) The mass fraction of the polysaccharide polymer in the mixed solution is 0.1%-5%; (5) The gradient crosslinking reaction temperature is 25℃-50℃, and the gradient crosslinking reaction time is 0.5h-48h; (6) The cleaning process includes: first, water washing; then, alcohol replacement; then, soaking in an aqueous solution of 0.2%-1% anionic descaling agent for 1-2 hours to swell; and finally, placing it in water. (7) The first matrix material and the second matrix material are independently selected from dermal decellularized matrix or collagen.

20. The use of a biological patch as described in any one of claims 1-11 in the repair of tissue damage.

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