Tissue sealing, isolating and repairing matrix and preparation method thereof

Abstract

The invention provides a tissue sealing, isolating and repairing matrix and a preparation method thereof, and belongs to the technical field of tissue matrixes. Comprising the following steps: (1) extracting collagen in animal tissues by an enzymolysis method; (2) dissolving with an acid solution or a phosphate buffer solution to prepare a collagen solution; (3) removing bubbles in the collagen solution by adopting centrifugation or vacuum; (4) spreading the defoamed collagen solution in a flat mold, and slowly dehydrating step by step to form a collagen film; (5) cross-linking by adopting a chemical agent or a physical method; (6) soaking and cleaning; (7) slowly removing water step by step to obtain a collagen matrix with a thickness of 5-30 [mu] m; and (8) slitting, packaging and sterilizing to prepare the tissue sealing, isolating and repairing matrix, which is extremely thin and extremely soft in texture, shows extremely strong adhesion and adaptability to the tissue boundary, can be spontaneously and tightly attached to the tissue surface, and effectively prevents the problems of air leakage, low-pressure seepage, errhysis and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue matrix, and specifically relates to a tissue sealing, isolating and repairing matrix and a preparation method thereof. Background Art

[0002] Currently, tissue sealing film products usually consist of a basement membrane and a bioadhesive. Although there are various types of adhesives, they all have certain biocompatibility problems. For example, cyanoacrylate adhesives have poor biodegradability and can cause tissue inflammatory reactions. NHS (N-hydroxysuccinimide) active ester is a bioadhesive with excellent performance, having 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 is that the NHS active ester group can react with the amino group of lysine in tissue proteins to form an amide bond, thereby adhering the basement membrane to the tissue surface. In the domestic market, tissuepatch of Tissuemed company is currently the only commercialized tissue sealing film product, adopting the technical scheme of a PLGA polymer thin film compounded with NHS active ester.

[0003] Chinese invention patent CN115814163B discloses a self-adhesive patch made of a collagen matrix and NHS active ester groups. However, active esters have problems such as high price and complex coating processes, which greatly increase production costs and time. In addition, the NHS group can only react with lysine, and parts such as the large intestine and small intestine (common parts where leakage often occurs after surgery) may be covered with a fat layer, which will hinder the contact between the NHS group and lysine in the tissue and affect the sealing effect of the product. Moreover, NHS active ester has strict requirements for the storage environment and needs to be stored at low temperature, increasing the costs of transportation and storage and posing a risk of product failure. These factors limit the wide clinical application of tissue sealing film products. Summary of the Invention

[0004] The purpose of the present invention is to provide a tissue sealing, isolating and repairing matrix and a preparation method thereof. Through a unique preparation process, a tissue sealing, isolating and repairing matrix with an extremely thin thickness and extremely soft texture is prepared, showing extremely strong conformability and adaptability to tissues, being able to spontaneously and tightly adhere to the tissue surface, and effectively preventing problems such as air leakage, low-pressure liquid leakage and blood leakage.

[0005] The technical solution of the present invention is realized as follows:

[0006] The present invention provides a preparation method of a tissue sealing, isolating and repairing matrix, including the following steps:

[0007] (1) Extract collagen from animal tissues by an enzymatic hydrolysis method;

[0008] (2) Dilute it with an acid solution or a phosphate buffer solution to a collagen solution with a concentration of 0.1 - 1%;

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

[0010] (4) Spread the degassed collagen solution evenly in a flat mold, and slowly dehydrate it step by step to form a collagen film;

[0011] (5) Crosslink by using a chemical agent or a physical method;

[0012] (6) Soak and wash;

[0013] (7) Slowly dehydrate it step by step to obtain a collagen matrix with a thickness of 5 - 30 μm;

[0014] (8) Cut, package, sterilize, and prepare a tissue sealing, isolating, and repairing matrix.

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

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

[0017] S1. Add ficin to a denaturant solution, stir and react, dialyze, and freeze-dry to obtain denatured intermediate-state ficin;

[0018] S2. Add the denatured intermediate-state ficin to an ionic liquid, perform ultra-high pressure treatment, remove the ionic liquid under reduced pressure, dissolve it in water, filter, and freeze-dry to obtain intermediate-state ficin;

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

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

[0021] The present invention prepares a modified ficin. Under the synergistic action of the denaturants guanidine hydrochloride and sodium dodecylbenzenesulfonate, at an appropriate concentration, the Tyr (tyrosine) and Trp (tryptophan) residues in the enzyme molecule change from the undissociated state in the natural state to the dissociated state, and subtle conformational changes occur locally in the molecule, thereby increasing the enzyme activity.

[0022] Adding the denatured intermediate state ficin into ionic liquid, the ionic liquid extraction technology presents advantages such as high extraction rate, stable properties, small solvent consumption, easy removal, and low pollution. At the same time, in the ionic liquid, the peptide chain of the denatured intermediate state ficin unfolds, and the buried Tyr (tyrosine) residues in the enzyme molecule are continuously exposed, resulting in an increase in enzyme activity. Under the treatment of ultra-high pressure, the secondary structure of the enzyme peptide chain molecule changes from a helical structure to a β-sheet structure mainly, thus rapidly increasing the enzyme activity, obtaining highly active ficin, significantly improving the enzymatic hydrolysis efficiency of the substrate animal tissue, reducing the enzymatic hydrolysis time, avoiding the breakage of collagen molecular chains caused by long-term high-temperature treatment, maintaining the integrity of collagen molecular chains, enhancing the flexibility and adhesiveness of the product, and thus improving the tissue sealing effect.

[0023] In addition, in the present invention, the prepared intermediate state ficin is coupled to a magnetic iron oxide carrier modified with polydopamine. On the one hand, it increases the specific surface area of the modified enzyme, can enhance the interaction between the enzyme and the substrate, improve the immobilization efficiency and stability of the enzyme, and at the same time can endow the enzyme with better thermal stability and pH stability, also improving the enzyme catalytic efficiency and shortening the action time.

[0024] As a further improvement of the present invention, in step S1, the denaturing agent solution is a PBS buffer solution containing 3-5 wt% guanidine hydrochloride and 1-2 wt% sodium dodecylbenzenesulfonate, the pH value of the PBS buffer solution is 7.3-7.6, the temperature of the stirring reaction is room temperature, and the time is 10-15 h; in step S2, 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 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, in step S3, the pH value of the Tris-HCl solution is 8.5-9.5, the mass ratio of the magnetic iron oxide to dopamine hydrochloride is 10:3-6, the temperature of the heating and stirring reaction is 50-60 °C, and the time is 3-5 h; in step S4, the mass ratio of the intermediate state ficin, NHS, EDC, and modified iron oxide is 3-5:1-2:1-2:12-15, the time of the stirring activation is 20-40 min, and the time of the stirring reaction is 10-15 h.

[0026] As a further improvement of the present invention, the animal tissue is at least one of bovine Achilles tendon, pigskin, 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 solution is 7.4 - 7.7.

[0028] As a further improvement of the present invention, the chemical agent is an amine group, imine group or aldehyde group crosslinking agent; the physical method is ultraviolet lamp irradiation.

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

[0030] The present invention further protects a tissue sealing, isolating and repairing matrix prepared by the above preparation method.

[0031] The present invention further protects the application of a tissue sealing, isolating and repairing matrix prepared by the above preparation method in the field of tissue sealing.

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

[0033] Through a unique preparation process, the present invention prepares a tissue sealing, isolating and repairing matrix with an extremely thin thickness and extremely soft texture, showing extremely strong conformability and adaptability to tissues. It can spontaneously and tightly adhere to the tissue surface, effectively preventing problems such as air leakage, low-pressure liquid leakage and blood leakage.

[0034] Different from common tissue sealing membranes, the ultra-thin tissue sealing, isolating and repairing matrix of the present invention can achieve tissue sealing without adding adhesives, avoiding potential risks and adverse reactions caused by adhesives. Moreover, the preparation process of this product is simple, greatly reducing the production cost and improving the cost performance of the product.

[0035] In terms of clinical use, for traditional common sealing membrane products, pressing is required at the action site during use to promote sufficient contact and adhesion between the adhesive and the tissue, and this process takes a long time. However, the tissue sealing, isolating and repairing matrix of the present invention is extremely convenient to use. Just gently attach it to the required part, without pressing and waiting, it can quickly achieve tight fitting of the tissue, shortening the operation time and improving the clinical operation efficiency. In addition, different from the specificity of the action site of the adhesive, the tissue sealing, isolating and repairing matrix of the present invention breaks through the application limitations of traditional sealing membranes and can be used in non-protein tissue parts, significantly broadening the scope of application of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0037] Figure 1 It is a comparison graph of the degradation experiment in Example 1;

[0038] Figure 2 It is the structure diagrams of Example 1 of the present invention and Bio-Gide. A is the SEM electron micrograph of the product prepared in Example 1, and B is the structure diagram of Bio-Gide (from the product promotional materials);

[0039] Figure 3 It is the sealing effect diagram of the animal tissue in Example 4. Specific implementation manners

[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

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

[0042] Example 1

[0043] First, using 100 g of bovine Achilles tendon as the raw material, adding 250 mL of 1 wt% ficin aqueous solution, enzymatically hydrolyzing at 37 °C for 24 hours, filtering and washing to obtain collagen.

[0044] Second, dissolving the collagen with 0.3 wt% malonic acid solution to a concentration of 0.5 wt%;

[0045] Third, removing the bubbles in the collagen solution by vacuum pumping;

[0046] Fourth, spreading 150 g of the collagen solution on a flat mold, and slowly dehydrating step by step to form a collagen film;

[0047] Fifth, crosslinking with glutaraldehyde solution;

[0048] Sixth, soaking and washing.

[0049] Seventh, slowly dehydrating step by step to obtain a collagen matrix with a thickness of about 5 μm.

[0050] Eighth, cutting, packaging and sterilizing to obtain a tissue sealing, isolating and repairing matrix.

[0051] Example 2

[0052] It is different from Example 1 in that by adjusting the amount of the collagen solution, the thickness of the tissue sealing, isolating and repairing matrix prepared is about 10 μm.

[0053] Example 3

[0054] It is different from Example 1 in that by adjusting the amount of the collagen solution, the thickness of the tissue sealing, isolating and repairing matrix prepared is about 20 μm.

[0055] Example 4

[0056] It is different from Example 1 in that by adjusting the amount of the collagen solution, the thickness of the tissue sealing, isolating and repairing matrix prepared is about 30 μm.

[0057] Comparative Example 1

[0058] It is different from Example 1 in that by adjusting the amount of the collagen solution, the thickness of the tissue sealing, isolating and repairing matrix prepared is about 50 μm.

[0059] Comparative Example 2

[0060] It is different from Example 1 in that by adjusting the amount of the collagen solution, the thickness of the tissue sealing, isolating and repairing matrix prepared is about 75 μm.

[0061] Comparative Example 3

[0062] It is different from Example 1 in that by adjusting the amount of the collagen solution, the thickness of the tissue sealing, isolating and repairing matrix prepared is about 100 μm.

[0063] Comparative Example 4

[0064] It is different from Example 1 in that by adjusting the amount of the collagen solution, the thickness of the tissue sealing, isolating and repairing matrix prepared is about 200 μm.

[0065] Preparation Example 1

[0066] The method is as follows:

[0067] S1. Add 1 g of ficin to 50 mL of a PBS buffer solution with a pH of 7.3 containing 3 wt% of guanidine hydrochloride and 1 wt% of sodium dodecylbenzenesulfonate, stir and react at room temperature for 10 h, dialyze using a dialysis bag with a pore size of 10 kDa, and lyophilize the non-permeated liquid to obtain denatured intermediate-state ficin;

[0068] S2. Add 1 g of denatured intermediate state ficin into 50 mL of 1-decyl-3-methylimidazolium bromide, perform ultra-high pressure treatment at 200 MPa for 20 min, remove 1-decyl-3-methylimidazolium bromide under reduced pressure, dissolve with water, filter, and freeze-dry to obtain intermediate state ficin;

[0069] S3. Add 1 g of magnetic iron oxide into 50 mL of Tris-HCl solution with a pH value of 8.5, add 0.3 g of dopamine hydrochloride, heat to 50 °C, stir and react for 3 h, separate with a magnet, wash, and dry to obtain modified iron oxide;

[0070] S4. Add 0.3 g of intermediate state ficin into 50 mL of water, add 0.1 g of NHS and 0.1 g of EDC, stir and activate at 0 °C for 20 min, add 1.2 g of modified iron oxide, stir and react for 10 h, separate with a magnet, wash, and dry to obtain modified ficin.

[0071] Preparation Example 2

[0072] The method is as follows:

[0073] S1. Add 1 g of ficin into 50 mL of PBS buffer solution with a pH value of 7.3 - 7.6 containing 5 wt% hydrochloric acid guanidine and 2 wt% sodium dodecylbenzenesulfonate, stir and react at room temperature for 15 h, dialyze with a dialysis bag with a pore size of 15 kDa, and freeze-dry the non-permeated liquid to obtain denatured intermediate state ficin;

[0074] S2. Add 1 g of denatured intermediate state ficin into 50 mL of 1-octyl-3-methylimidazolium bromide, perform ultra-high pressure treatment at 300 MPa for 40 min, remove 1-octyl-3-methylimidazolium bromide under reduced pressure, dissolve with water, filter, and freeze-dry to obtain intermediate state ficin;

[0075] S3. Add 1 g of magnetic iron oxide into 50 mL of Tris-HCl solution with a pH value of 9.5, add 0.6 g of dopamine hydrochloride, heat to 60 °C, stir and react for 5 h, separate with a magnet, wash, and dry to obtain modified iron oxide;

[0076] S4. Add 0.5 g of intermediate state ficin into 50 mL of water, add 0.2 g of NHS and 0.2 g of EDC, stir and activate at 0 °C for 40 min, add 1.5 g of modified iron oxide, stir and react for 15 h, separate with a magnet, wash, and dry to obtain modified ficin.

[0077] Preparation Example 3

[0078] The method is as follows:

[0079] S1. Add 1 g of ficin to 50 mL of PBS buffer solution with a pH of 7.4 containing 4 wt% guanidine hydrochloride and 1.2 wt% sodium dodecylbenzenesulfonate, stir and react at room temperature for 12 h, dialyze using a dialysis bag with a pore size of 2 kDa, and lyophilize the non-permeated liquid to obtain denatured intermediate-state ficin;

[0080] S2. Add 1 g of denatured intermediate-state ficin to 50 mL of 1-butyl-3-methylimidazolium bromide, perform ultra-high pressure treatment at 250 MPa for 30 min, remove 1-butyl-3-methylimidazolium bromide under reduced pressure, dissolve in water, filter, and lyophilize to obtain intermediate-state ficin;

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

[0082] S4. Add 0.4 g of intermediate-state ficin to 50 mL of water, add 0.15 g of NHS and 0.15 g of EDC, stir and activate at 0 °C for 30 min, add 1.3 g of modified iron oxide, stir and react for 12 h, separate with a magnet, wash, and dry to obtain modified ficin.

[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. Add 1 g of ficin to 50 mL of PBS buffer solution with a pH of 7.4 containing 5.2 wt% sodium dodecylbenzenesulfonate, stir and react at room temperature for 12 h, dialyze using a dialysis bag with a pore size of 2 kDa, and lyophilize the non-permeated liquid to obtain denatured intermediate-state ficin.

[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. Add 1 g of ficin to 50 mL of PBS buffer solution with a pH of 7.4 containing 5.2 wt% guanidine hydrochloride, stir and react at room temperature for 12 h, dialyze using a dialysis bag with a pore size of 2 kDa, and lyophilize the non-permeated liquid to obtain denatured intermediate-state ficin.

[0091] Comparative Preparation Example 3

[0092] It is different from Preparation Example 3 in that step S1 is not carried out.

[0093] Specifically as follows:

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

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

[0096] S3. Add 0.4 g of intermediate-state ficin to 50 mL of water, add 0.15 g of NHS and 0.15 g of EDC, stir and activate at 0 °C for 30 min, add 1.3 g of modified iron tetroxide, stir and react for 12 h, separate with a magnet, wash, and dry to obtain modified ficin.

[0097] Comparative Preparation Example 4

[0098] It is different from Preparation Example 3 in that 1-butyl-3-methylimidazolium bromide in step S2 is replaced by PBS buffer solution with pH = 7.4.

[0099] Specifically as follows:

[0100] S2. Add 1 g of denatured intermediate-state ficin to 50 mL of PBS buffer solution with pH = 7.4, perform ultra-high pressure treatment at 250 MPa for 30 min, filter, dialyze, and freeze-dry the non-permeated liquid to obtain intermediate-state ficin.

[0101] Comparative Preparation Example 5

[0102] It is different from Preparation Example 3 in that the ultra-high pressure treatment is not carried out in step S2.

[0103] Specifically as follows:

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

[0105] Comparative Preparation Example 6

[0106] It is different from Preparation Example 3 in that step S2 is not carried out.

[0107] The details are as follows:

[0108] S1. Add 1 g of ficin to 50 mL of PBS buffer solution with a pH of 7.4 containing 4 wt% hydrochloric acid guanidine and 1.2 wt% sodium dodecylbenzenesulfonate, stir and react at room temperature for 12 h, dialyze using a dialysis bag with a pore size of 2 kDa, and lyophilize the non-permeated solution to obtain denatured intermediate-state ficin;

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

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

[0111] Comparative Preparation Example 7

[0112] The difference from Preparation Example 3 is that steps S1 and S2 are not carried out.

[0113] The details are as follows:

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

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

[0116] Example 5

[0117] The difference from Example 4 is that the ficin is replaced by the modified ficin prepared in Preparation Example 1 with the same mass, and enzymolysis is carried out at 37 °C for 2 h. The thickness of the prepared tissue sealing, isolation, and repair matrix is about 30 μm.

[0118] Example 6

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

[0120] Example

[0121] The difference from Example 5 is that the modified ficin is prepared from Preparation Example 3.

[0122] Comparative Example 5

[0123] The difference from Example 5 is that the modified ficin is prepared from Comparative Preparation Example 1.

[0124] Comparative Example 6

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

[0126] Comparative Example 7

[0127] The difference from Example 5 is that the modified ficin is prepared from Comparative Preparation Example 3.

[0128] Comparative Example 8

[0129] The difference from Example 5 is that the modified ficin is prepared from Comparative Preparation Example 4.

[0130] Comparative Example 9

[0131] The difference from Example 5 is that the modified ficin is prepared from Comparative Preparation Example 5.

[0132] Comparative Example 10

[0133] The difference from Example 5 is that the modified ficin is prepared from Comparative Preparation Example 6.

[0134] Comparative Example 11

[0135] The difference from Example 5 is that the modified ficin is prepared from Comparative Preparation Example 7. [[ID=Z46]]

[0136] Test Example 1 Performance Test

[0137] The tissue sealing, isolating and repairing matrices prepared in Examples 1-7 and Comparative Examples 1-11 and the commercially available tissue sealing film product Tissuepatch were subjected to performance tests.

[0138] (1) Shear strength

[0139] Refer to the method in YY / T 0729.1-2009, cut the fresh pigskin graft and the sample into strips with a width of 2.5 cm, lap the sample with the pigskin graft, and the lap area is 2.5 cm × 1.0 cm. After the sample and the pigskin graft are closely attached, measure the shear strength. Clamp both ends of the specimen on the fixture of the tensile testing machine, make the loading direction be the long axis direction of the specimen, and load the specimen at a speed of 5 mm / min until the lap area separates, record the maximum force and calculate the shear strength.

[0140] (2) Compressive property

[0141] Refer to ASTM F2392-04, take a fresh pig stomach, make a circular notch with a diameter of 5 mm on the surface, cut the sample into a size of 2.5 cm × 2.5 cm, and evenly attach it to the defect in a centrally symmetric manner. Connect one end of the sample to a pressure gauge and introduce gas at the other end to measure the maximum pressure that the defect can withstand.

[0142] Table 1

[0143]

[0144]

[0145] Shear strength and compressive property are the key indicators to measure the performance of tissue adhesives. Judging from the data of each example, as the product thickness gradually decreases, both the shear strength and compressive property of the product show an upward trend. Products with too large thickness (Comparative Examples 1-4) have poor flexibility and adhesiveness, and their shear strength and compressive property cannot meet the requirements. When the product thickness decreases from about 50 μm (Comparative Example 1) to about 30 μm (Example 4), the shear strength and compressive property are significantly improved, achieving unexpected results. After testing, the shear strength of the commercial tissue sealing film product tissuepatch is 13.5 Kpa, and the compressive property is 2.3 KPa. There is no significant difference in performance between Example 4 and the commercial product. Therefore, the performance of the product in Example 4 meets the clinical use requirements. The reason may be that as the product thickness continues to decrease, the flexibility and adhesiveness of the product break through the critical point, so that it can be closely attached to the tissue. When the product thickness continues to decrease, the compressive property and shear strength of the product will also increase accordingly. The compressive property of Example 1 reaches 5.1 kPa, and the shear strength reaches 26.2 kPa. Because in the actual production process, when the thickness decreases to about 5 μm, the product yield is already relatively low, and it is no longer practically feasible to further reduce the thickness.

[0146] The products of Examples 5-7 have the same thickness as the product of Example 4, but there are significant improvements in shear strength and compressive properties, achieving a more excellent tissue sealing effect. The reason is that the modified ficin improves the efficiency of collagen enzymolysis, and collagen extraction can be completed in a shorter time (reduced from 24 hours to 2 hours), avoiding the breakage of collagen molecular chains caused by long-term high-temperature treatment and maintaining its integrity. The integrity of the molecular chain is likely to be the core factor affecting the flexibility and adhesion of the product. In addition, the modified ficin can be separated by a magnet and thus reused, which can significantly reduce the production cost.

[0147] Test Example 2 In vitro cytotoxicity

[0148] In vitro cytotoxicity is an important indicator to measure the biocompatibility of products. This test is carried out based on the product per unit area. Since the declared product is extremely thin, the substances causing cytotoxicity in the unit area should be significantly reduced compared with conventional products. Therefore, it should have certain advantages in terms of cytotoxicity. The self-adhesive biological patch substrate material disclosed in Patent CN115814163B is the same as the product of the present invention, both are collagen. However, since this product has not been commercialized, samples cannot be obtained for comparison. The Bio-Gide absorbable biofilm (Geistlich Pharma AG, Switzerland) has been commercialized for many years and is widely used in the fields of tissue isolation and repair. The raw material of this product is also collagen, so the comparison with this product is typical.

[0149] Take the tissue sealing, isolation and repair matrices prepared in Examples 1-7, Comparative Example 4, and the commercially available Tissuepatch and Bio-Gide absorbable biofilm, and conduct tests in accordance with GB / T 16886.5-2017. The higher the cell survival rate, the smaller the cytotoxicity. The results are shown in Table 2.

[0150] Table 2

[0151] Group Cell survival rate (%) 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 absorbable biofilm 81.56

[0152] From the results analysis of each example and Comparative Example 4, it is found that as the thickness of the product decreases, the cell survival rate gradually increases. Combining the influence of the product thickness on the tissue sealing effect, it shows that the reduction of the product thickness not only improves the tissue sealing effect but also enhances the biocompatibility level. For in vivo implants, it represents a double improvement in clinical safety and effectiveness.

[0153] After comparing each example with the Bio-Gide absorbable biofilm, it is found that the product of this patent is significantly superior to this commercially available collagen tissue repair product in terms of cell survival rate. Since the raw materials are both collagen, the thickness of the Bio-Gide absorbable biofilm is about 0.3 mm. The product of this patent may show better biocompatibility due to its extremely thin thickness.

[0154] In addition, compared with Tissuepatch, each embodiment has a lower cytotoxicity level, indicating that the patented product has better biocompatibility than existing commercial tissue sealing membrane products. The thickness of Tissuepatch is about 50μm, which further corroborates that the extremely thin characteristic of the product may be the key factor for its excellent biocompatibility.

[0155] Test Example 3 Degradation Performance

[0156] The degradation time of the absorbable tissue repair material plays an important role in the clinical effect. If the degradation time is too short, the material may lose its function prematurely, affecting the effects of isolation and tissue repair. Due to the extremely thin thickness of the product, to verify whether its degradation performance can meet the clinical use requirements of tissue repair materials, we carried out a comparative study on the degradation performance between Example 1 and the commercial collagen tissue repair product Bio-Gide absorbable biofilm.

[0157] Both Example 1 and the commercially available Bio-Gide absorbable biofilm were cut into 1 cm × 1 cm splines, added to the collagenase solution for in vitro degradation tests. Using a 37°C water bath, samples were taken out at 3 h, 6 h, 9 h, 12 h, 24 h, 30 h, and 36 h of degradation, dried and weighed, and the residual rate of the samples was calculated.

[0158] The results showed (see Figure 1 ), that the Bio-Gide absorbable biofilm was basically completely degraded after 24 hours, while Example 1 required 36 h for complete degradation, showing stronger anti-degradation performance than the commercial product. Therefore, it can meet the clinical requirements for the degradation time of tissue repair materials. This may be due to the unique preparation process of the patented product and the dense structure of the product (see Figure 2 A), making it difficult for the collagenase solution to penetrate and exchange quickly, thus delaying the degradation process. Although the Bio-Gide absorbable biofilm has an advantage in thickness (about 0.3 mm), its structure is loose and porous (see Figure 2 B), enabling the collagenase solution to penetrate and exchange quickly, accelerating the degradation rate of the material.

[0159] Test Example 4 Animal Tissue Sealing Effect

[0160] To evaluate the sealing performance of the product on tissue wounds in animals, 3 New Zealand rabbits were selected as experimental subjects. The experimental animals were deeply anesthetized, the surgical site was shaved, disinfected, covered with a surgical drape and fixed. A longitudinal incision about 10 cm long was made along the midline of the abdomen in the middle part to expose the liver. One lobe of the liver was fixed, a small incision about 5-8 mm was made with a scalpel, hemostasis was applied by pressing for several seconds, and then non-absorbable sutures were used for rapid suture. Then, hemostasis was continued by pressing until a small amount of blood / effusion still flowed out. The surface of the organs within 5 cm around the wound was wiped dry to make the surrounding area relatively dry without liquid. The product of Example 4 was cut into a size of about 3*3 cm and applied to the wound. After observing for 5 minutes, if there was no bleeding or effusion, the liver was carefully replaced back to its original position in the abdominal cavity. The stomach test was the same as that of the liver. After the test was completed, the skin wound was closed and disinfected after suture. After raising the animals for seven days, the animals were sacrificed and it was observed whether the product adhered firmly to the wound.

[0161] The results are as Figure 3 , during the surgical operation process, within 5 minutes after the experimental product was applied, there was no bleeding or effusion on the wound surface, indicating that the product can effectively prevent blood leakage from the tissue wound and has a good sealing effect. On the 7th day after the operation, through anatomical observation, it was found that the product still adhered firmly to the wound surface. Since the normal healing cycle of animal tissue wounds is usually about 7 days, it shows that the product can continuously play a sealing role during the entire wound healing period, effectively preventing the occurrence of effusion phenomenon and meeting the basic requirements of clinical application.

[0162] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a matrix for tissue sealing, isolation and repair, characterized in that, It includes the following steps: (1) Extract collagen from animal tissues by enzymatic hydrolysis method; (2) Dilute it with an acid solution or a phosphate buffer solution to a collagen solution of 0.1-1%; (3) Remove the air bubbles in the collagen solution by centrifugation or vacuum; (4) Spread the degassed collagen solution on a flat mold, and slowly dehydrate it step by step to form a collagen film; (5) Crosslink by chemical agents or physical methods; (6) Immerse and wash; (7) Slowly dehydrate it step by step to obtain a collagen matrix with a thickness of 5-30 μm; (8) Cut, package, sterilize, and prepare a tissue sealing, isolation, and repair matrix.

2. The preparation method according to claim 1, characterized in that, The enzyme used in the enzymatic hydrolysis is ficin or modified ficin.

3. The preparation method according to claim 1, characterized in that, The preparation method of the modified ficin is as follows: S1. Add ficin to a denaturing agent solution, stir and react, dialyze, and freeze-dry to obtain denatured intermediate-state ficin; S2. Add the denatured intermediate-state ficin to an ionic liquid, perform ultra-high pressure treatment, remove the ionic liquid under reduced pressure, dissolve it in water, filter, and freeze-dry to obtain intermediate-state ficin; S3. Add magnetic iron oxide to a Tris-HCl solution, add dopamine hydrochloride, heat and stir to react, separate with a magnet, wash, and dry to obtain modified iron oxide; S4. Add the intermediate-state ficin to water, add NHS and EDC, stir and activate, add the modified iron oxide, stir and react, separate with a magnet, wash, and dry to obtain modified ficin.

4. The preparation method according to claim 1, characterized in that, In step S1, the denaturing agent solution is a PBS buffer solution containing 3-5 wt% guanidine hydrochloride and 1-2 wt% sodium dodecylbenzenesulfonate, the pH value of the PBS buffer solution is 7.3-7.6, the temperature of the stirring reaction is room temperature, and the time is 10-15 h; in step S2, 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 pressure of the ultra-high pressure treatment is 200-300 MPa, and the time is 20-40 min.

5. The preparation method according to claim 1, characterized in that, In step S3, the pH value of the Tris-HCl solution is 8.5-9.5, the mass ratio of the magnetic iron oxide to dopamine hydrochloride is 10:3-6, the temperature of the heating and stirring reaction is 50-60 °C, and the time is 3-5 h; in step S4, the mass ratio of the intermediate-state ficin, NHS, EDC, and the modified iron oxide is 3-5:1-2:1-2:12-15, the time of the stirring activation is 20-40 min, and the time of the stirring reaction is 10-15 h.

6. The preparation method according to claim 1, wherein, The animal tissue is at least one of bovine tendon, pig's trotters, and fish skin.

7. 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 solution is 7.4-7.

7.

8. The preparation method according to claim 1, wherein The chemical agent is an amine group, imine group, or aldehyde group crosslinking agent; the physical method is ultraviolet lamp irradiation; the aldehyde group crosslinking agent is glutaraldehyde.

9. A tissue sealing, isolating and repairing matrix prepared by the preparation method according to any one of claims 1-8.

10. Use of a tissue sealing, isolating and repairing matrix prepared by the preparation method according to any one of claims 1-8 in the field of tissue sealing.

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