A tissue sealing, isolating and repair matrix and method of making same

Abstract

The application provides a tissue sealing, isolating and repairing matrix and a preparation method thereof, and belongs to the technical field of tissue matrixes.The matrix comprises the following steps: (1) extracting collagen in animal tissue by an enzymatic hydrolysis method; (2) dissolving and preparing a collagen solution by using an acid solution or a phosphate buffer solution; (3) removing air bubbles in the collagen solution by centrifugation or vacuum; (4) laying the defoamed collagen solution in a flat mold, and forming a collagen film after slow and step-by-step dehydration; (5) crosslinking by using a chemical agent or a physical method; (6) soaking and cleaning; (7) slowly and step-by-step removing water to obtain a collagen matrix with a thickness of 5-30 microns; and (8) cutting and packaging, and sterilizing to obtain the tissue sealing, isolating and repairing matrix, which is extremely thin and soft, has strong adhesion and adaptability to a tissue interface, can spontaneously and tightly adhere to a tissue surface, and can effectively prevent air leakage, low-pressure liquid seepage and blood seepage and the like.

Description

Technical Field

[0001] This invention relates to the field of tissue matrix technology, specifically to a tissue sealing, isolation and repair matrix and its preparation method. Background Technology

[0002] Currently, tissue sealing membrane products typically consist of a basement membrane and a bio-adhesive. While adhesives are diverse, they all present certain biocompatibility issues. For example, cyanoacrylate adhesives have poor biodegradability and can cause tissue inflammation. NHS (N-hydroxysuccinimide) active ester is a high-performance bio-adhesive with advantages such as low toxicity and specific adhesion to tissue proteins, and has been widely used in the field of tissue sealing in recent years. Its mechanism of action involves the NHS active ester groups reacting with the amino groups of lysine in tissue proteins to form amide bonds, thereby adhering the basement membrane to the tissue surface. In the domestic market, Tissuemed's tissuepatch is currently the only commercially available tissue sealing membrane product, employing a PLGA polymer film composite with NHS active ester technology.

[0003] Chinese invention patent CN115814163B discloses a self-adhesive patch based on a collagen matrix and NHS active ester groups. However, active esters suffer from high prices and complex coating processes, significantly increasing production costs and time. Furthermore, NHS groups can only react with lysine, and areas such as the large and small intestines (common sites where postoperative leakage is common) may be covered by a fat layer, hindering the contact between the NHS groups and lysine in the tissue and affecting the product's sealing effect. Moreover, NHS active esters require stringent storage conditions, necessitating low-temperature storage, increasing transportation and storage costs and posing a risk of product failure. These factors limit the widespread clinical application of tissue sealing film products. Summary of the Invention

[0004] The purpose of this invention is to propose a tissue sealing, isolation and repair matrix and its preparation method. Through a unique preparation process, an extremely thin and extremely soft tissue sealing, isolation and repair matrix is ​​prepared, which exhibits strong adhesion and adaptability to the tissue interface. It can spontaneously and tightly adhere to the tissue surface and effectively prevent problems such as air leakage, low-pressure fluid leakage and bleeding.

[0005] The technical solution of this invention is implemented as follows:

[0006] This invention provides a method for preparing a tissue sealing, isolation, and repair matrix, comprising the following steps:

[0007] (1) Extraction of collagen from animal tissues by enzymatic hydrolysis;

[0008] (2) Dilute the collagen solution to 0.1-1% using acid solution or phosphate buffer;

[0009] (3) Remove air bubbles from the collagen solution by centrifugation or vacuum;

[0010] (4) The dehydrated collagen solution is spread in a flat mold and slowly dehydrated to form a collagen film.

[0011] (5) Crosslinking is performed using chemical agents or physical methods;

[0012] (6) Soaking and washing;

[0013] (7) Remove water slowly and gradually to obtain a collagen matrix with a thickness of 5-30 μm;

[0014] (8) Cut, package, sterilize, and obtain tissue sealing, isolation and repair matrix.

[0015] As a further improvement of the present invention, the enzyme used in the enzymatic hydrolysis is fig protease or modified fig protease.

[0016] As a further improvement of the present invention, the preparation method of the modified fig protease is as follows:

[0017] S1. Add fig protease to a denaturing agent solution, stir and react, dialyze, freeze dry, and obtain denatured intermediate fig protease;

[0018] S2. The denatured intermediate fig protease was added to an ionic liquid, subjected to ultra-high pressure treatment, the ionic liquid was removed by depressurization, dissolved in water, filtered, and freeze-dried to obtain the intermediate fig protease.

[0019] S3. Add magnetic iron oxide to Tris-HCl solution, add dopamine hydrochloride, heat and stir to react, separate with magnets, wash, dry, and obtain modified iron oxide;

[0020] S4. Add intermediate fig protease to water, add NHS and EDC, stir to activate, add modified iron oxide, stir to react, separate with a magnet, wash, dry, and obtain modified fig protease.

[0021] This invention prepares a modified fig protease. Under the synergistic effect of the exfoliant guanidine hydrochloride and sodium dodecylbenzenesulfonate at appropriate concentrations, the Tyr (tyrosine) and Trp (tryptophan) residues in the enzyme molecule change from their native non-dissociated state to a dissociated state, resulting in subtle conformational changes in the local molecule, thereby improving enzyme activity.

[0022] Adding denatured intermediate fig protease to ionic liquids offers advantages such as high extraction rate, stable properties, low solvent consumption, easy removal, and low pollution. Simultaneously, in ionic liquids, the denatured intermediate fig protease peptide chains unwind, continuously exposing the embedded Tyr (tyrosine) residues within the enzyme molecule, thus enhancing enzyme activity. Under ultra-high pressure treatment, the secondary structure of the enzyme peptide chain changes from predominantly helical to predominantly β-sheet, resulting in a rapid increase in enzyme activity and the acquisition of highly active fig protease. This significantly improves the enzymatic hydrolysis efficiency on animal tissue substrates, reduces hydrolysis time, avoids collagen chain breakage caused by prolonged high-temperature treatment, maintains the integrity of collagen molecular chains, and enhances the product's flexibility and adhesion, thereby improving tissue sealing effects.

[0023] In addition, the present invention couples the prepared intermediate fig protease to a polydopamine-modified magnetic iron oxide support, which on the one hand increases the specific surface area of ​​the modified enzyme, enhances the interaction between the enzyme and the substrate, improves the enzyme's immobilization efficiency and stability, and also endows the enzyme with better thermal stability and pH stability, thereby improving the enzyme's catalytic efficiency and shortening the reaction time.

[0024] As a further improvement of the present invention, the denaturing agent solution in step S1 is a PBS buffer solution containing 3-5 wt% guanidine hydrochloride and 1-2 wt% sodium dodecylbenzenesulfonate, wherein the pH value of the PBS buffer solution is 7.3-7.6, the stirring reaction temperature is room temperature, and the time is 10-15 h; the ionic liquid in step S2 is selected from at least one of 1-ethyl-3-methylimidazolium bromide, 1-butyl-3-methylimidazolium bromide, 1-octyl-3-methylimidazolium bromide, 1-decyl-3-methylimidazolium bromide, and 1-butyl-3-methylimidazolium chloride, wherein the pressure of the ultra-high pressure treatment is 200-300 MPa, and the time is 20-40 min.

[0025] As a further improvement of the present invention, the pH value of the Tris-HCl solution in step S3 is 8.5-9.5, the mass ratio of the magnetic iron oxide and dopamine hydrochloride is 10:3-6, and the heating and stirring reaction time is 50-60℃ for 3-5 hours; in step S4, the mass ratio of the intermediate fig protease, NHS, EDC and modified iron oxide is 3-5:1-2:1-2:12-15, the stirring activation time is 20-40 minutes, and the stirring reaction time is 10-15 hours.

[0026] As a further improvement of the present invention, the animal tissue is at least one of bovine Achilles tendon, pig skin, and fish skin.

[0027] As a further improvement of the present invention, the acid solution is a malonic acid solution, and the pH value of the phosphate buffer is 7.4-7.7.

[0028] As a further improvement of the present invention, the chemical agent is an amino-, imine-, or aldehyde-based crosslinking agent; the physical method is ultraviolet light irradiation.

[0029] As a further improvement of the present invention, the aldehyde crosslinking agent is glutaraldehyde.

[0030] The present invention further protects a tissue sealing, isolation and repair matrix prepared by the above-described preparation method.

[0031] This invention further protects the application of a tissue sealing, isolation and repair matrix prepared by the above-described method in the field of tissue sealing.

[0032] The present invention has the following beneficial effects:

[0033] This invention utilizes a unique preparation process to produce an extremely thin and soft tissue sealing, isolation, and repair matrix that exhibits strong adhesion and adaptability to the tissue interface. It can spontaneously and tightly adhere to the tissue surface, effectively preventing problems such as air leakage, low-pressure fluid seepage, and bleeding.

[0034] Unlike common tissue sealing films, the ultrathin tissue sealing, isolation, and repair matrix of this invention achieves tissue sealing without the need for adhesives, avoiding the potential risks and adverse reactions associated with adhesives. Furthermore, the product's manufacturing process is simple, significantly reducing production costs and improving its cost-effectiveness.

[0035] In clinical applications, traditional sealing film products require pressing at the application site to promote full contact and adhesion between the adhesive and tissue, a time-consuming process. In contrast, the tissue sealing, isolation, and repair matrix of this invention is extremely convenient to use. Simply apply it gently to the desired area; no pressing or waiting is required, and it quickly achieves close tissue adhesion, shortening surgical time and improving clinical efficiency. Furthermore, unlike the specificity of adhesive application sites, the tissue sealing, isolation, and repair matrix of this invention overcomes the application limitations of traditional sealing films, allowing its use in non-protein tissue sites and significantly broadening the product's applicability. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention 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 of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This is a comparison diagram of the degradation experiment in Example 1;

[0038] Figure 2 The diagrams are of Embodiment 1 and Bio-Gide structure. A is an SEM image of the product prepared in Embodiment 1, and B is a Bio-gide structure diagram (from product promotional materials).

[0039] Figure 3 This is a diagram showing the sealing effect of animal tissue in Example 4. Detailed Implementation

[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Fig protease; magnetic iron oxide with an average particle size of 500 nm; NHS, N-hydroxysuccinimide; EDC, 1-ethyl-(3-dimethylaminopropyl)carbodiimide.

[0042] Example 1

[0043] 1. Using 100g of beef Achilles tendon as raw material, add 250mL of 1wt% fig protease aqueous solution, enzymatically hydrolyze at 37℃ for 24 hours, filter and wash to obtain collagen.

[0044] 2. Dissolve collagen in 0.3wt% malonic acid solution to a concentration of 0.5wt%;

[0045] 3. Vacuum extraction is used to remove air bubbles from the collagen solution;

[0046] 4. Spread 150g of collagen solution evenly in a flat mold, and slowly dehydrate it step by step to form a collagen film.

[0047] 5. Crosslinking using glutaraldehyde solution;

[0048] 6. Soaking and washing.

[0049] 7. The water is slowly removed step by step to obtain a collagen matrix with a thickness of about 5μm.

[0050] 8. Cut, package, and sterilize to obtain a tissue sealing, isolation, and repair matrix.

[0051] Example 2

[0052] The difference from Example 1 is that, by adjusting the amount of collagen solution, the thickness of the tissue sealing, isolation and repair matrix is ​​approximately 10 μm.

[0053] Example 3

[0054] The difference from Example 1 is that, by adjusting the amount of collagen solution, the thickness of the tissue sealing, isolation and repair matrix is ​​approximately 20 μm.

[0055] Example 4

[0056] The difference from Example 1 is that, by adjusting the amount of collagen solution, the thickness of the tissue sealing, isolation and repair matrix is ​​approximately 30 μm.

[0057] Comparative Example 1

[0058] The difference from Example 1 is that, by adjusting the amount of collagen solution, the thickness of the tissue sealing, isolation and repair matrix is ​​approximately 50 μm.

[0059] Comparative Example 2

[0060] The difference from Example 1 is that, by adjusting the amount of collagen solution, the thickness of the tissue sealing, isolation and repair matrix is ​​approximately 75 μm.

[0061] Comparative Example 3

[0062] The difference from Example 1 is that, by adjusting the amount of collagen solution, the thickness of the tissue sealing, isolation and repair matrix is ​​approximately 100 μm.

[0063] Comparative Example 4

[0064] The difference from Example 1 is that, by adjusting the amount of collagen solution, the thickness of the tissue sealing, isolation and repair matrix is ​​approximately 200 μm.

[0065] Preparation Example 1

[0066] The method is as follows:

[0067] S1. 1g of fig protease was added to 50mL of PBS buffer solution with pH 7.3 containing 3wt% guanidine hydrochloride and 1wt% sodium dodecylbenzenesulfonate. The mixture was stirred at room temperature for 10h and dialyzed using a dialysis bag with a pore size of 10kDa. The unpermeated solution was freeze-dried to obtain denatured intermediate fig protease.

[0068] S2. Add 1g of denatured intermediate fig protease to 50mL of 1-decyl-3-methylimidazolium bromide, treat with ultra-high pressure at 200MPa for 20min, remove 1-decyl-3-methylimidazolium bromide under reduced pressure, dissolve in water, filter, freeze dry to obtain intermediate fig protease.

[0069] S3. Add 1g of magnetic iron oxide to 50mL of Tris-HCl solution with pH 8.5, add 0.3g of dopamine hydrochloride, heat to 50℃, stir for 3h, separate the magnets, wash, dry, and obtain modified iron oxide.

[0070] S4. Add 0.3g of intermediate fig protease to 50mL of water, add 0.1g of NHS and 0.1g of EDC, stir and activate at 0℃ for 20min, add 1.2g of modified iron oxide, stir and react for 10h, separate by magnet, wash, dry, and obtain modified fig protease.

[0071] Preparation Example 2

[0072] The method is as follows:

[0073] S1. 1g of fig protease was added to 50mL of PBS buffer solution with pH 7.3-7.6 containing 5wt% guanidine hydrochloride and 2wt% sodium dodecylbenzenesulfonate. The mixture was stirred at room temperature for 15h and dialyzed using a 15kDa dialysis bag. The unpermeated solution was freeze-dried to obtain denatured intermediate fig protease.

[0074] S2. Add 1g of denatured intermediate fig protease to 50mL of 1-octyl-3-methylimidazolium bromide, treat with ultra-high pressure at 300MPa for 40min, remove 1-octyl-3-methylimidazolium bromide under reduced pressure, dissolve in water, filter, freeze dry to obtain intermediate fig protease.

[0075] S3. Add 1g of magnetic iron oxide to 50mL of Tris-HCl solution with pH 9.5, add 0.6g of dopamine hydrochloride, heat to 60℃, stir for 5h, separate the magnets, wash, dry, and obtain modified iron oxide.

[0076] S4. Add 0.5g of intermediate fig protease to 50mL of water, add 0.2g of NHS and 0.2g of EDC, stir and activate at 0℃ for 40min, add 1.5g of modified iron oxide, stir and react for 15h, separate by magnet, wash, dry, and obtain modified fig protease.

[0077] Preparation Example 3

[0078] The method is as follows:

[0079] S1. 1g of fig protease was added to 50mL of PBS buffer solution with pH 7.4 containing 4wt% guanidine hydrochloride and 1.2wt% sodium dodecylbenzenesulfonate. The mixture was stirred at room temperature for 12h and dialyzed using a dialysis bag with a pore size of 2kDa. The unpermeated solution was freeze-dried to obtain denatured intermediate fig protease.

[0080] S2. Add 1g of denatured intermediate fig protease to 50mL of 1-butyl-3-methylimidazolium bromide, treat with ultra-high pressure at 250MPa for 30min, remove 1-butyl-3-methylimidazolium bromide under reduced pressure, dissolve in water, filter, freeze dry to obtain intermediate fig protease.

[0081] S3. Add 1g of magnetic iron oxide to 50mL of Tris-HCl solution with pH 9, add 0.45g of dopamine hydrochloride, heat to 55℃, stir and react for 4h, separate the magnets, wash, dry, and obtain modified iron oxide.

[0082] S4. Add 0.4g of intermediate fig protease to 50mL of water, add 0.15g of NHS and 0.15g of EDC, stir and activate at 0℃ for 30min, add 1.3g of modified iron oxide, stir and react for 12h, separate by magnet, wash, dry, and obtain modified fig protease.

[0083] Comparative Preparation Example 1

[0084] The difference from Preparation Example 3 is that guanidine hydrochloride was not added in step S1.

[0085] Specifically as follows:

[0086] S1. 1g of fig protease was added to 50mL of PBS buffer solution with pH 7.4 containing 5.2wt% sodium dodecylbenzenesulfonate. The mixture was stirred at room temperature for 12h and dialyzed using a dialysis bag with a pore size of 2kDa. The undiluted solution was freeze-dried to obtain denatured intermediate fig protease.

[0087] Comparative Preparation Example 2

[0088] The difference from Preparation Example 3 is that sodium dodecylbenzenesulfonate was not added in step S1.

[0089] Specifically as follows:

[0090] S1. 1g of fig protease was added to 50mL of PBS buffer solution with pH 7.4 containing 5.2wt% guanidine hydrochloride. The mixture was stirred at room temperature for 12h and dialyzed using a dialysis bag with a pore size of 2kDa. The undiluted solution was freeze-dried to obtain denatured intermediate fig protease.

[0091] Comparative preparation example 3

[0092] The difference from Preparation Example 3 is that step S1 was not performed.

[0093] Specifically as follows:

[0094] S1. Add 1g of fig protease to 50mL of 1-butyl-3-methylimidazolium bromide, treat with ultra-high pressure at 250MPa for 30min, remove 1-butyl-3-methylimidazolium bromide under reduced pressure, dissolve in water, filter, freeze dry to obtain intermediate fig protease.

[0095] S2. Add 1g of magnetic iron oxide to 50mL of Tris-HCl solution with pH 9, add 0.45g of dopamine hydrochloride, heat to 55℃, stir and react for 4h, separate the magnets, wash, dry, and obtain modified iron oxide.

[0096] S3. Add 0.4g of intermediate fig protease to 50mL of water, add 0.15g of NHS and 0.15g of EDC, stir and activate at 0℃ for 30min, add 1.3g of modified iron oxide, stir and react for 12h, separate by magnet, wash, dry, and obtain modified fig protease.

[0097] Comparative preparation example 4

[0098] The difference from Preparation Example 3 is that in step S2, 1-butyl-3-methylimidazolium bromide is replaced with PBS buffer at pH 7.4.

[0099] Specifically as follows:

[0100] S2. Add 1g of denatured intermediate fig protease to 50mL of PBS buffer (pH=7.4), autoclave at 250MPa for 30min, filter, dialyze, and freeze-dry the impurity to obtain intermediate fig protease.

[0101] Comparative preparation example 5

[0102] The difference from Preparation Example 3 is that no ultra-high pressure treatment was performed in step S2.

[0103] Specifically as follows:

[0104] S2. Add 1g of denatured intermediate fig protease to 50mL of 1-butyl-3-methylimidazolium bromide, stir for 30min, remove 1-butyl-3-methylimidazolium bromide under reduced pressure, dissolve in water, filter, freeze dry to obtain intermediate fig protease.

[0105] Comparative preparation example 6

[0106] The difference from Preparation Example 3 is that step S2 was not performed.

[0107] Specifically as follows:

[0108] S1. 1g of fig protease was added to 50mL of PBS buffer solution with pH 7.4 containing 4wt% guanidine hydrochloride and 1.2wt% sodium dodecylbenzenesulfonate. The mixture was stirred at room temperature for 12h and dialyzed using a dialysis bag with a pore size of 2kDa. The unpermeated solution was freeze-dried to obtain denatured intermediate fig protease.

[0109] S2. Add 1g of magnetic iron oxide to 50mL of Tris-HCl solution with pH 9, add 0.45g of dopamine hydrochloride, heat to 55℃, stir and react for 4h, separate the magnets, wash, dry, and obtain modified iron oxide.

[0110] S3. Add 0.4g of denatured intermediate fig protease to 50mL of water, add 0.15g of NHS and 0.15g of EDC, stir and activate at 0℃ for 30min, add 1.3g of modified iron oxide, stir and react for 12h, separate by magnet, wash, dry, and obtain modified fig protease.

[0111] Comparative preparation example 7

[0112] The difference from Preparation Example 3 is that steps S1 and S2 were not performed.

[0113] Specifically as follows:

[0114] S1. Add 1g of magnetic iron oxide to 50mL of Tris-HCl solution with pH 9, add 0.45g of dopamine hydrochloride, heat to 55℃, stir and react for 4h, separate the magnets, wash, dry, and obtain modified iron oxide.

[0115] S2. Add 0.4g of fig protease to 50mL of water, add 0.15g of NHS and 0.15g of EDC, stir and activate at 0℃ for 30min, add 1.3g of modified iron oxide, stir and react for 12h, separate by magnet, wash, dry, and obtain modified fig protease.

[0116] Example 5

[0117] The difference from Example 4 is that the fig protease was replaced by an equal mass of the modified fig protease prepared in Preparation Example 1, and the mixture was enzymatically hydrolyzed at 37°C for 2 hours. The resulting tissue sealing, isolation, and repair matrix had a thickness of approximately 30 μm.

[0118] Example 6

[0119] The difference from Example 5 is that the modified fig protease was prepared in Preparation Example 2.

[0120] Example 7

[0121] The difference from Example 5 is that the modified fig protease was prepared in Preparation Example 3.

[0122] Comparative Example 5

[0123] The difference from Example 5 is that the modified fig protease was prepared by Comparative Preparation Example 1.

[0124] Comparative Example 6

[0125] The difference from Example 5 is that the modified fig protease was prepared from Comparative Preparation Example 2.

[0126] Comparative Example 7

[0127] The difference from Example 5 is that the modified fig protease was prepared by Comparative Preparation Example 3.

[0128] Comparative Example 8

[0129] The difference from Example 5 is that the modified fig protease was prepared by Comparative Preparation Example 4.

[0130] Comparative Example 9

[0131] The difference from Example 5 is that the modified fig protease was prepared by Comparative Preparation Example 5.

[0132] Comparative Example 10

[0133] The difference from Example 5 is that the modified fig protease was prepared by Comparative Preparation Example 6.

[0134] Comparative Example 11

[0135] The difference from Example 5 is that the modified fig protease was prepared by Comparative Preparation Example 7.

[0136] Test Example 1 Performance Test

[0137] The tissue sealing, isolation, and repair matrices prepared in Examples 1-7 and Comparative Examples 1-11, as well as the commercially available tissue sealing membrane product Tissuepatch, were subjected to performance tests.

[0138] (1) Shear strength

[0139] Referring to the method in YY / T 0729.1-2009, fresh pigskin grafts and samples were cut into strips 2.5 cm wide. The samples were then overlapped with the pigskin grafts, with an overlap area of ​​2.5 cm × 1.0 cm. After the samples and pigskin grafts were tightly bonded, the shear strength was measured. Both ends of the sample were clamped in the tensile testing machine fixtures, with the force direction along the long axis of the sample. The sample was loaded at a speed of 5 mm / min until the overlap area separated. The maximum force was recorded, and the shear strength was calculated.

[0140] (2) Compressive strength

[0141] Referring to ASTM F2392-04, take a fresh pig stomach and make a circular notch with a diameter of 5mm on the surface. Cut the sample into 2.5cm×2.5cm pieces and apply them flat to the defect in a centrally symmetrical manner. Connect a pressure gauge to one end of the sample and introduce gas into the other end to measure the maximum pressure that the defect can withstand.

[0142] Table 1

[0143]

[0144]

[0145] Shear strength and compressive strength are key indicators for evaluating the performance of tissue adhesives. Data from various examples show that both shear strength and compressive strength increase as product thickness decreases. Products with excessive thickness (Comparative Examples 1-4) fail to meet requirements for shear strength and compressive strength due to poor flexibility and adhesion. However, when the product thickness decreases from approximately 50 μm (Comparative Example 1) to approximately 30 μm (Example 4), shear strength and compressive strength significantly improve, achieving unexpected results. Testing showed that the commercially available tissue patch product has a shear strength of 13.5 kPa and a compressive strength of 2.3 kPa. Example 4 showed no significant difference in performance compared to the commercial product; therefore, the performance of Example 4 meets clinical requirements. This may be because as the product thickness continues to decrease, its flexibility and adhesion exceed a critical point, allowing for a tighter fit with the tissue. As the product thickness continues to decrease, the compressive strength and shear strength also increase, with Example 1 achieving a compressive strength of 5.1 kPa and a shear strength of 26.2 kPa. In actual production, when the thickness is reduced to around 5µm, the product yield is already low, and further reducing the thickness is no longer practically feasible.

[0146] The products in Examples 5-7 have the same thickness as those in Example 4, but exhibit significantly improved shear strength and compressive strength, achieving a superior tissue sealing effect. This is because the modified fig protease enhances the efficiency of collagen hydrolysis, enabling collagen extraction in a shorter time (reduced from 24 hours to 2 hours). This avoids the breakage of collagen molecular chains caused by prolonged high-temperature treatment, maintaining their integrity. The integrity of the molecular chains is likely a core factor affecting the product's flexibility and adhesion. Furthermore, the modified fig protease can be separated using a magnet for reuse, significantly reducing production costs.

[0147] Test Example 2: In vitro cytotoxicity

[0148] In vitro cytotoxicity is an important indicator for evaluating the biocompatibility of a product. This test is based on the product per unit area. Because the product submitted for approval is extremely thin, the amount of cytotoxic substances per unit area should be significantly reduced compared to conventional products, thus giving it a certain advantage in cytotoxicity. The self-adhesive bio-patch base material disclosed in patent CN115814163B is the same as the product of this invention, both being collagen. However, since this product has not yet been commercialized, samples cannot be obtained for comparison. Bio-Gide absorbable biomembrane (Gesseres, Switzerland) has been commercialized for many years and is widely used in tissue isolation and repair. This product also uses collagen as its raw material, making the comparison with it typical.

[0149] The tissue sealing, isolation, and repair matrices prepared in Examples 1-7 and Comparative Example 4, as well as commercially available Tissuepatch and Bio-gide absorbable biomembranes, were tested according to GB / T 16886.5-2017. Higher cell viability indicates lower cytotoxicity. The results are shown in Table 2.

[0150] Table 2

[0151] Group Cell viability (%) Blank control 100.00 negative control 101.22 Positive control 4.85 Example 1 99.89 Example 2 97.57 Example 3 94.48 Example 4 92.35 Example 5 93.44 Example 6 92.25 Example 7 93.10 Comparative Example 4 78.24 Tissuepatch 83.09 Bio-gide can absorb biomembranes 81.56

[0152] Analysis of the results from various embodiments and Comparative Example 4 revealed that cell viability gradually increased as the product thickness decreased. Considering the impact of product thickness on tissue sealing, this indicates that reducing product thickness improves both tissue sealing and biocompatibility. For implantable devices, this represents a dual improvement in clinical safety and efficacy.

[0153] Comparison of the various embodiments with the Bio-gide absorbable biomembrane revealed that this patented product significantly outperforms this commercially available collagen tissue repair product in terms of cell survival rate. Since the raw material is the same collagen, and the Bio-gide absorbable biomembrane is approximately 0.3 mm thick, this patented product may exhibit superior biocompatibility due to its extremely thin thickness.

[0154] Furthermore, the embodiments exhibited lower levels of cytotoxicity compared to Tissuepatch, indicating that this patented product has better biocompatibility than existing commercial tissue sealing membrane products. The Tissuepatch's thickness of approximately 50 μm further supports the conclusion that its extremely thin profile is likely a key factor in its superior biocompatibility.

[0155] Test Example 3 Degradation Performance

[0156] The degradation time of absorbable tissue repair materials plays a crucial role in clinical efficacy. If the degradation time is too short, the material may lose its function prematurely, affecting its isolation and tissue repair effects. Due to the extremely thin thickness of the product, to verify whether its degradation performance meets the clinical requirements for tissue repair materials, we conducted a comparative study on the degradation performance of Example 1 and the commercially available collagen tissue repair product, Bio-Gide absorbable biomembrane.

[0157] Both Example 1 and the commercially available Bio-Gide absorbable biomembrane were cut into 1cm×1cm strips, and collagenase solution was added for in vitro degradation tests. The samples were taken out at 37°C water bath, 3h, 6h, 9h, 12h, 24h, 30h and 36h after degradation, dried and weighed, and the sample residue rate was calculated.

[0158] The results show (see) Figure 1 Bio-Gide absorbable biofilms are essentially completely degraded after 24 hours, while Example 1 requires 36 hours for complete degradation, demonstrating stronger anti-degradation performance than commercial products. Therefore, it meets clinical requirements regarding the degradation time of tissue repair materials. This may be attributed to the unique manufacturing process of the patented product and its dense structure (see...). Figure 2 A) This makes it difficult for collagenase solution to penetrate and exchange rapidly, thus slowing down the degradation process. Although Bio-Gide absorbable biomembranes have an advantage in thickness (approximately 0.3 mm), their structure is loose and porous (see [reference needed]). Figure 2 B) enables the collagenase solution to rapidly penetrate and exchange, accelerating the degradation rate of the material.

[0159] Test Example 4: Sealing Effect of Animal Tissue

[0160] To evaluate the sealing performance of the product on tissue wounds in animals, three New Zealand rabbits were selected as experimental subjects. The animals were deeply anesthetized, and the surgical area was shaved, disinfected, draped, and secured. A longitudinal incision of approximately 10 cm was made along the midline of the abdomen to expose the liver. One lobe of the liver was secured, and a small incision of approximately 5-8 mm was made using a scalpel. Pressure was applied for hemostasis for several seconds, and the incision was quickly sutured with non-absorbable sutures. Pressure was then applied again until a small amount of blood / exudate continued to flow. The surface of organs within a 5 cm radius of the wound was wiped dry to ensure a relatively dry environment. A piece of the product from Example 4, approximately 3*3 cm in size, was cut and applied to the wound. After 5 minutes of observation, if no bleeding or exudate was observed, the liver was carefully returned to its original position in the abdominal cavity. The stomach test was performed in the same manner as the liver test. After the test, the skin wound was closed, sutured, and disinfected. Seven days later, the animals were euthanized, and the adhesion of the product to the wound was observed.

[0161] The results are as follows Figure 3 During the surgical procedure, no bleeding or exudation occurred on the wound within 5 minutes of application of the experimental product, indicating that the product effectively prevents blood seepage from the wound and provides a good seal. On the 7th day post-surgery, anatomical observation revealed that the product remained firmly attached to the wound surface. Since the normal healing period for animal wounds is typically about 7 days, this demonstrates that the product can continuously provide a seal throughout the entire wound healing process, effectively preventing exudation and meeting the basic requirements for clinical application.

[0162] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a tissue sealing, isolation, and repair matrix, characterized in that, Includes the following steps: (1) Extraction of collagen from animal tissues by enzymatic hydrolysis; (2) Dilute the collagen solution to 0.1-1% using acid solution or phosphate buffer; (3) Remove air bubbles from the collagen solution by centrifugation or vacuum; (4) The dehydrated collagen solution is spread in a flat mold and slowly dehydrated to form a collagen film. (5) Crosslinking using chemical agents or physical methods; (6) Soaking and washing; (7) The water is slowly removed step by step to obtain a collagen matrix with a thickness of 5-30 μm; (8) Cut, package, sterilize, and prepare tissue sealing, isolation and repair matrix; The enzyme used in the enzymatic hydrolysis is modified fig protease; The modified fig protease is prepared as follows: S1. Fig protease is added to a denaturing agent solution and stirred to react, dialyzed, and freeze-dried to obtain denatured intermediate fig protease; the denaturing agent solution is a PBS buffer solution containing 3-5 wt% guanidine hydrochloride and 1-2 wt% sodium dodecylbenzenesulfonate, the pH of the PBS buffer solution is 7.3-7.6, the stirring reaction is carried out at room temperature for 10-15 h; S2. The denatured intermediate fig protease is added to an ionic liquid, subjected to ultra-high pressure treatment, the ionic liquid is removed under reduced pressure, dissolved in water, filtered, and freeze-dried to obtain the intermediate fig protease; the ionic liquid is selected from at least one of 1-ethyl-3-methylimidazolium bromide, 1-butyl-3-methylimidazolium bromide, 1-octyl-3-methylimidazolium bromide, 1-decyl-3-methylimidazolium bromide, and 1-butyl-3-methylimidazolium chloride; the ultra-high pressure treatment is performed at a pressure of 200-300 MPa for 20-40 min; S3. Magnetic iron oxide (Fe3O4) is added to a Tris-HCl solution, dopamine hydrochloride is added, the mixture is heated and stirred, separated by magnets, washed, and dried to obtain modified iron oxide (Fe3O4); the pH value of the Tris-HCl solution is 8.5-9.5, the mass ratio of magnetic iron oxide (Fe3O4) to dopamine hydrochloride is 10:3-6, and the heating and stirring reaction time is 50-60℃ for 3-5 hours. S4. Add intermediate fig protease to water, add NHS and EDC, stir to activate, add modified iron oxide, stir to react, separate with a magnet, wash, dry, and obtain modified fig protease. The mass ratio of the intermediate fig protease, NHS, EDC and modified iron oxide is 3-5:1-2:1-2:12-15, the stirring activation time is 20-40 min, and the stirring reaction time is 10-15 h.

2. The preparation method according to claim 1, characterized in that, The animal tissue is at least one of the following: bovine Achilles tendon, pig's trotter, and fish skin.

3. The preparation method according to claim 1, characterized in that, The acid solution is a malonic acid solution, and the pH value of the phosphate buffer is 7.4-7.

7.

4. The preparation method according to claim 1, characterized in that, The chemical agent is an amino, imino, or aldehyde crosslinking agent; the physical method is ultraviolet light irradiation; the aldehyde crosslinking agent is glutaraldehyde.

5. A tissue sealing, isolation, and repair matrix prepared by the preparation method according to any one of claims 1-4.

6. The application of a tissue sealing, isolation and repair matrix prepared by the preparation method according to any one of claims 1-4 in the field of tissue sealing.

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