Dura mater biological patch and preparation method thereof

Abstract

The invention of biological dura mater patch and a preparation method thereof. Raw material of patch does not come from commercial meat animals, but from breeding animals, such as SIS patch of the sow, has natural good mechanical properties; the method uses plant-derived reagents in the decellularization process, no damage to ECM natural structural base, and retain more active ingredients, has a better ability to induce tissue repair and growth; neither uses a cross-linking agent to enhance mechanical properties, nor synthetic detergents used to remove cells; chemical residues and their toxicity are avoided, while more effective ingredients in ECM, especially GAGs be retained. The patch has soft texture and good toughness, easy to sew tightly, and prevent cerebrospinal fluid leakage; and degradation rate is basically synchronized with the growth of the new dura mater, which is more conducive to repair and regeneration of dura mater.

Claims

1. A biological patch for repairing dura mater, wherein the patch contains decellularized connective tissue of breeding stock, the breeding stock is specifically adult, reproducible stock, kept for at least one year, excluding young reserve breeding stock, as contrasted to commercial meat stock.

2. The patch of claim 1, wherein the decellularizing agent is mainly composed of saponin.

3. The patch of claim 1, wherein the breeding stock includes sow, cow, ewe, and mare.

4. The patch of claim 1, wherein the connective tissue is one of, or a combination of any of: the submucosa of the small intestine, the submucosa of the bladder, the submucosa of the stomach, the dermal matrix, the pericardium, the meninges, the amniotic membrane, the visceral membrane, and the peritoneum.

5. The patch according to claim 1, wherein the connective tissue of the breeding stock is the submucosa of the small intestine of the sow.

6. A method for preparing the patch of claim 1, wherein the decellularization reagent in the decellularization process is mainly composed of plant-derived nonionic surfactant.

7. The method according to claim 6, wherein the decellularizing agent is one of plant-derived triterpenesaponins, steroid saponins, or combination thereof.

8. The method according to claim 6, wherein the decellularizing agent is one of Quil-A, tea saponin, or combination thereof.

9. The method according to claim 6, wherein the effective working concentration weight ratio of the decellularizing agent is 0.05-1%; the decellularizing agent acts on the patch multiple times, each action time being the action time with the patch material is 10-60 minutes, and action temperature being 4-15 C.

10. The method according to claim 6, wherein raw material of the method for preparing the dura mater is mainly composed of connective tissue of the breeding stock.

Description

DETAILED DESCRIPTION

Example 1

[0069] The specific steps for the preparation of porcine small intestinal submucosa dura mater are as follows:

[0070] 1) Material extraction and washing: pre-treatment: according to the breeding records, select the breeding days of about 24 months, the binary mixed sow in the empty stage, after slaughtering, clean the fresh small intestine tissue;

[0071] 2) Pretreatment: remove the mucosal layer, muscular layer, serous membrane layer, and lymph nodes of the pig small intestine by physical scraping, separate the submucosa, and soak in 0.5% acetic acid solution for 30 minutes. The ratio of pig small intestine to acetic acid solution is 1:5. Soak in purified water three times to obtain the raw material of the biological patch, namely the submucosa of the small intestine, hereinafter referred to as SIS material;

[0072] 3) Sterilization: Use a mixed aqueous solution containing 1.0% peroxyacetic acid and 15% ethanol. The ratio of SIS material to mixed aqueous solution is 1:10. Under ultrasonic conditions, immerse at room temperature for 100 minutes for disinfection. After that, use purified water for ultrasonic cleaning 3 times;

[0073] 4) Degreasing: use ethanol with a concentration of 90%, the ratio of SIS material to ethanol is 1:10, under ultrasonic conditions, soak at room temperature for 2 h; afterwards, use ultrasonic cleaning with water for injection 3 limes;

[0074] 5) Decellularization: use a solution containing 0.25% saponin (from Quil-A, the working concentration is calculated based on the content of pure saponin), soak the raw material of the patch at 4 C. and ultrasonic conditions for 30 minutes; then use the same concentration of 0.5 The saponin solution is used to rinse the patch material for 10 minutes; then the patch is soaked with PBS-EDTA solution for 20 minutes; the previous decellularization step is repeated once, and the total time is about 120 minutes;

[0075] 6) Remove DNA and remove -Gal antigen: use an aqueous solution containing SU/ml DNase, the ratio of SIS material to DNase solution is 1:5, soak for 20 minutes at 37 C. under ultrasound; then use PBS Rinse 3 times; use an aqueous solution containing SU/ml -galactosidase, the ratio of SIS material to -galactosidase solution is 1:5, soak for 20 minutes at 30 C. under ultrasound; then use PBS solution rinse;

[0076] 7) Use an aqueous solution of NaOH with a concentration of 10 mM, the ratio of SIS material to NaOH solution is 1:20, under ultrasonic conditions, soak for 50 minutes at room temperature; then use PBS ultrasonic cleaning until neutral;

[0077] 8) Stereotypes, freeze-drying and sterilization: the decellularized sheet-like raw materials are intersected horizontally and crosswise among the four pieces, and are fixed on the mold overlappingly. After freeze-drying, packaging, and finally irradiation sterilization.

Example 2

[0078] The steps of pretreatment of small intestine tissue, disinfection, defatting, decellularization, deDNA removal and -Gal antigen removal, lyophilization, sterilization, etc., are completely the same as in Example 1; the difference is only in the initial animal selection, in slaughter In the field, the fresh small intestine of the eliminated sow is selected as the raw material for the patch; after in-depth understanding, the eliminated binary sow usually has a gestational age of more than 8 births and weighs about 180 kg, which is empty; after retrospective investigation and understanding, the elimination Sows have been raised for more than 40 months.

Example 3

[0079] The steps of pretreatment of small intestinal tissue, disinfection, defatting, decellularization, deDNA and -Gal antigen removal, lyophilization, sterilization, etc. are same as in Example 1; the difference is only in the initial animal selection; the slaughter is selected On the farm, the fresh small intestine tissues of DuchangSanyuan groceries pigs weighing about 100 kg were washed and cleaned; it is known that the number of breeding days is 160-180 days.

Example 4

[0080] Animal selection, pretreatment of connective tissue, disinfection, defatting, decellularization, deDNA removal and -Gal antigen removal, lyophilization, sterilization and other steps are exactly the same as in Example 3; the difference is only the choice of decellularization reagent in the fourth step Above, in this embodiment, 0.25% SDS was used to replace 0.25% saponin in Embodiment 3.

Example 5

[0081] Animal selection, connective tissue pretreatment, disinfection, defatting, decellularization, deDNA removal and -Gal antigen removal, lyophilization, sterilization and other steps are same as in Example 1; the difference is only in the selection of decellularization reagents in the fourth step in this example, 0.5% tea saponin was used instead of 0.25% saponin in Example 1.

Example 6

[0082] Animal selection, connective tissue pretreatment, disinfection, defatting, decellularization, deDNA removal and -Gal antigen removal, lyophilization, sterilization and other steps are same as in Example 2; the difference is only in the selection of decellularization reagents, In this example, 0.5% teasaponin was used instead of 0.25% saponin in Example 1.

Example 7: Optical Observation and Effective Component Detection of Dura Mater

[0083] Optical Microscope Observation:

[0084] Method: fixed with formalin, embedded in paraffin, cut the patch in the example into thin slices, dewaxed with xylene, dehydrated with alcohol, stained with hematoxylin-eosin, and observed the residual cells and matrix fibers under the microscope structure.

[0085] Results: In all of the decellularized patches in the six examples, no cells and fragments were observed; collagen fibers were continuous and of varying thickness, but no obvious breakage was observed.

[0086] Quantitative detection methods of glycosaminoglycans (GAGs) include Dubious method, Carbazole method, Elson-Morgan method, ELISA and electrophoresis method.

[0087] For the patch samples prepared in 1-6 examples, the content of the important active component glycosaminoglycans (GAGs) was detected using a commercial ELISA kit; the pretreatment method was to use low-temperature grinding method for various patches After processing, the results are shown in Table 1:

TABLE-US-00001 Glycosaminoglycan Group Patch source and treatment methods GAGs (ug/mg) Example 1 Sow's SIS + 0.25% saponin 6.43 0.38 Example 2 Culled sowSIS + 0.25% saponin 5.85 0.63 Example 3 Commercial Hog SIS + 0.25% saponin 6.56 0.56 Example 4 Commodity Hog SIS + 0.25% SDS 3.65 0.67 Example 5 Sow'sSIS + 0.5% tea saponin 6.20 0.39 Example 6 Culled sow SIS + 0.5% tea saponin 5.68 0.54

Example 8: Detection of Mechanical Properties of Dura Mater

[0088] The tensile strength of the dura mater samples prepared in Examples 1-6 was tested.

[0089] Method: Cut the sample into a shape with a width of 10 mm in two directions; after cutting, the sample was placed in an environment with a relative humidity of 40%-60% and a temperature of 222 C. for 2 hours. The distance between the clamps is 25 mm. Fix the two ends of the sample on the chuck of the tensile tester and stretch at a speed of 100 mm/min. Record the maximum force value at break.

[0090] The results are shown in Table 2 below:

TABLE-US-00002 Toughness (tensile strength) Group Patch source and treatment methods Unit: N Example 1 Sow's SIS + 0.25% saponin 48 Example 2 Culled sow SIS + 0.25% saponin 53 Example 3 Commercial Hog SIS + 0.25% saponin 34 Example 4 Commodity Hog SIS + 0.25% SDS 29 Example 5 Sow's SIS + 0.5% tea saponin 48 Example 6 Culled sow SIS + 0.5% tea saponin 50

[0091] A person of ordinary skill in the art can make various simple changes, adjustments or combinations to the present invention according to the above description; therefore, without prejudice to the spirit of the claims of the present invention, certain details in the embodiments should not it constitutes a limitation to the present invention, and the present invention will take the scope defined by the appended claims as the protection scope.