Modified small intestinal submucosa as well as preparation method and application thereof

Abstract

The invention provides a modified small intestinal submucosa which is a small intestinal submucosa grafted by methacrylic anhydride, and the grafting rate of the methacrylic anhydride is 40-90%. After cross-linking and curing, the modified small intestinal submucosa material provided by the invention has excellent biocompatibility, degradability and mechanical properties, can effectively promote revascularization and regulate immunoreaction, and has a wide application prospect as a tissue repair material.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and in particular relates to a modified small intestinal submucosa, a preparation method and application thereof. Background Art

[0002] The small intestinal submucosa (SIS) is one of the components of the small intestine. It is composed of a layer of dense connective tissue, mainly containing type I and III collagen, and contains a variety of growth factors: such as transforming growth factor-β (TGF-β), basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The hydrogel prepared by SIS has low immunogenicity and does not affect the immune response of the receptor to bacteria and viruses; good cell compatibility, tissue compatibility and in vivo degradation ability; regulates immune function and promotes angiogenesis. Therefore, SIS hydrogel has been used in the repair of bone, cartilage, skin and other injuries.

[0003] However, as a biogenic material, SIS hydrogel has poor mechanical properties and unstable gelation properties. Therefore, how to further modify SIS hydrogel to improve its mechanical properties and enrich its application forms while retaining its excellent biocompatibility, degradability, and ability to promote angiogenesis is of great significance to broadening the application of SIS materials. Summary of the invention

[0004] The purpose of the present invention is to provide a modified small intestinal submucosa and a preparation method and use thereof.

[0005] The invention provides a modified small intestinal submucosal layer material, which is a small intestinal submucosal layer grafted with methacrylic anhydride, and the methacrylic anhydride grafting rate is 40-90%.

[0006] Preferably, the methacrylic anhydride grafting rate is 40 to 90%.

[0007] Preferably, the methacrylic anhydride grafting rate is 80%.

[0008] Preferably, it is prepared by reacting small intestinal submucosal layer with methacrylic anhydride in an alkaline aqueous solution.

[0009] Preferably, the molar ratio of the free amino groups in the small intestinal submucosal layer to methacrylic anhydride is 1:(5-100).

[0010] Preferably, the molar ratio of the free amino groups in the small intestinal submucosal layer to methacrylic anhydride is 1:20.

[0011] Preferably, the alkaline aqueous solution is an aqueous solution with a pH of 8-10.

[0012] Preferably, the reaction conditions are: 0-50° C. for 1-24 h.

[0013] Preferably, the reaction conditions are: 0-20° C. for 20-24 hours, 20-35° C. for 10-20 hours, and 35-50° C. for 1-10 hours.

[0014] Preferably, the amino content of the small intestinal submucosal layer is 400-800 μM.

[0015] Preferably, the small intestinal submucosa is the small intestinal submucosa that has been defatted, decellularized and digested.

[0016] The present invention also provides a method for preparing the modified small intestinal submucosal material, comprising the following steps:

[0017] (1) The submucosa of the small intestine dissolves in water at 35 to 50°C;

[0018] (2) adding methacrylic anhydride, adjusting the pH to 8-10, and reacting at 35-50° C. for 1-10 hours;

[0019] Or, comprising the steps of:

[0020] (i) The small intestinal submucosa dissolves in water at 20-35°C;

[0021] (ii) adding methacrylic anhydride, adjusting the pH to 8-10, and reacting at 25° C. for 10-20 hours;

[0022] Or, comprising the steps of:

[0023] (a) The small intestinal submucosa is dissolved in water at 0 to 4°C;

[0024] (b) adding methacrylic anhydride, adjusting the pH to 8-10, and reacting at 0-4°C for 20-24 hours.

[0025] Preferably, the method further comprises the following steps: adding 4 to 6 times the volume of water to terminate the reaction, dialysis, and freeze-drying.

[0026] The present invention also provides a composition, which comprises the modified small intestinal submucosal layer and an initiator, wherein the weight ratio of the modified small intestinal submucosal layer to the initiator is 10-200:1.

[0027] Preferably, the weight ratio of the modified small intestinal submucosa to the initiator is 20-100:1.

[0028] Preferably, the initiator is a photoinitiator.

[0029] Preferably, the photoinitiator is selected from at least one of LAP or I2959.

[0030] Preferably, it also includes a solvent.

[0031] Preferably, the solvent is water.

[0032] The present invention also provides use of the composition for preparing gel.

[0033] The invention also provides a gel, which is prepared by reacting the above composition under light conditions.

[0034] Preferably, the wavelength of the illumination condition is 365-405 nm.

[0035] Preferably, in the composition, the initiator includes LAP, and the wavelength of the illumination condition is 405 nm;

[0036] Or, in the composition, the initiator includes I2959, and the wavelength of the illumination condition is 365 nm.

[0037] The invention also provides a tissue engineering scaffold obtained by freeze-drying the gel.

[0038] The present invention also provides the use of the gel or tissue engineering scaffold as a tissue repair material.

[0039] Beneficial effects of the present invention: After cross-linking and curing, the modified small intestinal submucosal layer material provided by the present invention has excellent biocompatibility, degradability and mechanical properties, can effectively promote angiogenesis, and has broad application prospects as a tissue repair material. In addition, the preparation method of the modified small intestinal submucosal layer of the present invention can be carried out at room temperature without the assistance of other cross-linking agents. Compared with the existing preparation methods, it has the advantages of lower energy consumption, simpler methods, and higher retention rate of bioactive components.

[0040] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0041] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1This is the relationship between the raw material ratio and the grafting rate of the modified small intestinal submucosal layer in Experimental Example 1.

[0043] Figure 2 For the determination of rheological properties of various hydrogels.

[0044] Figure 3 To determine the cytokine content in various hydrogels.

[0045] Figure 4 Comparison of the SISMA gelling properties obtained by two preparation methods. DETAILED DESCRIPTION

[0046] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0047] The small intestinal submucosal layer raw material used in the present invention is prepared by subjecting the isolated small intestinal submucosal layer to disinfection, degreasing, decellularization and descaling treatments in any order.

[0048] As an example, the following preparation method is provided:

[0049] 1) Take fresh pig small intestine, rinse the contents with water, rub it with salt and rinse it with water three times. Use a scalpel to cut the small intestine open and cut it into 10-20 cm long segments;

[0050] 2) Use a tongue depressor to scrape the mucosal layer, muscular layer and serosa layer of the small intestine and store in normal saline at 4°C;

[0051] 3) Rinse with deionized water, filter and immerse in a mixture of chloroform and methanol (chloroform: methanol = 1:1) at room temperature (25°C) overnight;

[0052] 4) After repeated washing with deionized water, place in 0.25% trypsin solution and treat at 4°C overnight;

[0053] 5) rinse with deionized water 10 times and then treat with 0.5% SDS for at least 4 h;

[0054] 6) After washing, freeze-dry at -70°C for 24 hours to obtain the SIS membrane.

[0055] 7) The SIS film is crushed using a ball mill at low temperature to obtain SIS powder.

[0056] 8) Digest the SIS powder with pepsin solution and freeze-dry it again for later use.

[0057] Example 1. Preparation of modified small intestinal submucosa

[0058] (1) Dissolve SIS in ultrapure water at a ratio of 1%, raise the temperature to 50°C, continue stirring, and add NaOH to adjust the pH to 9.

[0059] (2) After adding methacrylic anhydride (MA) dropwise in batches, NaOH was added to adjust the pH to a range of 8 to 9. The molar ratio of free amino groups (from SIS) to MA was 1:20, and the reaction was carried out at 50°C for a total reaction time of 3 hours. Dialysis and freeze-drying were performed for later use. In this embodiment, the method for calculating the amount of SIS is as follows: first, OPA was used to determine the free amino group content of SIS after digestion, and the actual amount of SIS was calculated based on this.

[0060] Example 2: Preparation of modified small intestinal submucosa

[0061] Dissolve SIS in an ice-water bath at a ratio of 0.25% for 4-6 hours. After SIS is dissolved, add free amino groups and methacrylic anhydride (MA) at a molar ratio of 1:20, adjust the pH value to 9-10, and react in an ice-water bath for 24 hours. Dialyze and freeze-dry for later use.

[0062] Example 3: Preparation of modified small intestinal submucosa

[0063] Dissolve SIS at a ratio of 0.5% at room temperature, add free amino groups and methacrylic anhydride (MA) at a molar ratio of 1:20, adjust the pH value to 9-10, and react at 25°C for 12 hours. Dialyze and freeze-dry for later use.

[0064] Example 4. Preparation of modified small intestinal submucosa

[0065] (1) Dissolve SIS in ultrapure water at a ratio of 1%, keep stirring, and after it is fully dissolved, add NaOH to adjust the pH to 9.

[0066] (2) After adding methacrylic anhydride (MA) dropwise in batches, NaOH was added to adjust the pH to a range of 8 to 9. The molar ratio of free amino groups (from SIS) to MA was 1:20, and a mixed solution was obtained by ultrasonic treatment (ultrasonic power 100 W, 4°C, 3h). Dialysis and freeze-drying were used for later use. In this embodiment, the method for calculating the amount of MA is as follows: first, the free amino group content after SIS digestion is determined by the OPA method, and based on this, the actual amount of MA is deduced according to the amount of free amino substances: the amount of MA substances is equal to 1:20.

[0067] Example 5. Composition for preparing gel and preparation of gel

[0068] The composition used to prepare the gel in this embodiment is as follows:

[0069] 5 g of the modified small intestinal submucosal material prepared in Example 1,

[0070] Photoinitiator LAP 0.25g,

[0071] 100g water.

[0072] The preparation method of the gel is:

[0073] After the above composition is uniformly mixed, a reaction is initiated under light conditions to obtain the product, wherein the wavelength of the light conditions is 405 nm.

[0074] The resulting gel can be freeze-dried and made into a tissue engineering scaffold for tissue repair.

[0075] Example 6. Composition for preparing gel and preparation of gel

[0076] The composition used to prepare the gel in this embodiment is as follows:

[0077] 10 g of the modified small intestinal submucosal material prepared in Example 2,

[0078] Photoinitiator I2959 0.5g,

[0079] 100g water.

[0080] The preparation method of the gel is:

[0081] After the above composition is uniformly mixed, a reaction is initiated under light conditions to obtain the product, wherein the wavelength of the light conditions is 365 nm.

[0082] The resulting gel can be freeze-dried and made into a tissue engineering scaffold for tissue repair.

[0083] Example 7. Composition for preparing gel and preparation of gel

[0084] The composition used to prepare the gel in this embodiment is as follows:

[0085] 3 g of the modified small intestinal submucosal material prepared in Example 1,

[0086] Photoinitiator LAP 0.1g,

[0087] 100g water.

[0088] The preparation method of the gel is:

[0089] After the above composition is uniformly mixed, a reaction is initiated under light conditions to obtain the product, wherein the wavelength of the light conditions is 405 nm.

[0090] The resulting gel can be freeze-dried and made into a tissue engineering scaffold for tissue repair.

[0091] Example 8. Composition for preparing gel and preparation of gel

[0092] The composition used to prepare the gel in this embodiment is as follows:

[0093] 15 g of the modified small intestinal submucosal material prepared in Example 2,

[0094] Photoinitiator LAP 0.25g,

[0095] 100g water.

[0096] The preparation method of the gel is:

[0097] After the above composition is uniformly mixed, a reaction is initiated under light conditions to obtain the product, wherein the wavelength of the light conditions is 405 nm.

[0098] The resulting gel can be freeze-dried and made into a tissue engineering scaffold for tissue repair.

[0099] Comparative Example 1SIS hydrogel

[0100] SIS 5g after digestion

[0101] Water 100g

[0102] The preparation method of the gel is:

[0103] (1) Separation of small intestinal submucosal layer: Wash fresh pig small intestine, cut it open along the long axis, cut it into 10-15 cm intestinal segments, mechanically scrape off the mucosal layer, muscular layer and serosa layer, and wash it repeatedly with PBS.

[0104] (2) Degreasing: Remove excess water, soak in degreasing solution at room temperature for 12 hours, take out and wash thoroughly.

[0105] (3) Decellularization: Soak the defatted intestinal segments in a pancreatic enzyme solution at 4°C for 12 hours, remove and wash, and then soak in an SDS solution at room temperature for 4 hours, remove and wash thoroughly.

[0106] (4) Freeze-drying and pulverization: The treated hindgut segments are vacuum dried and ground using a ball mill to prepare SIS powder.

[0107] (5) Digestion and re-freeze-drying: SIS powder is digested with pepsin solution and vacuum-freeze-dried again for later use.

[0108] (6) After dissolving SIS in water, adjust the pH to neutral, place at 37°C for 30 minutes, and then take out to form a hydrogel.

[0109] Comparative Example 2 GelMA hydrogel

[0110] GelMA 5g

[0111] Photoinitiator LAP 0.25g

[0112] 100g water

[0113] The preparation method of the gel is:

[0114] (1) Dissolve gelatin at a ratio of 0.5% in an ice-water bath for 4 to 6 hours. After it is completely dissolved, add free amino groups and methacrylic anhydride (MA) at a molar ratio of 1:20, adjust the pH value to 9 to 10, and react in an ice-water bath for 24 hours. Dialyze and freeze-dry for later use.

[0115] (2) After the above composition is uniformly mixed, a reaction is initiated under light conditions to obtain a product having a wavelength of 405 nm.

[0116] Comparative Example 3

[0117] SISMA 5g

[0118] Photoinitiator LAP 0.25g

[0119] 100g water

[0120] The preparation method of the gel is:

[0121] (1) Separation of small intestinal submucosal layer: Wash fresh pig small intestine, cut it open along the long axis, cut it into 10-15 cm intestinal segments, mechanically scrape off the mucosal layer, muscular layer and serosa layer, and wash it repeatedly with PBS.

[0122] (2) Degreasing: Remove excess water, soak in degreasing solution at room temperature for 12 hours, take out and wash thoroughly.

[0123] (3) Decellularization: Soak the defatted intestinal segments in a pancreatic enzyme solution at 4°C for 12 hours, remove and wash, and then soak in an SDS solution at room temperature for 4 hours, remove and wash thoroughly.

[0124] (4) Freeze-drying and pulverization: The treated hindgut segments are vacuum dried and ground using a ball mill to prepare SIS powder.

[0125] (5) Digestion and re-freeze-drying: SIS powder is digested with pepsin solution and vacuum-freeze-dried again for later use.

[0126] (6) Dissolve the digested SIS at a ratio of 0.5% for 4-6 hours. After it is completely dissolved, add the activated methacrylic acid, EDC and NHS mixture (free amino group: methacrylic acid: EDC: NHS molar ratio of 1:1:1:1, dissolved in N,N-dimethylformamide (DMF)) and react at room temperature (25°C) for 24 hours. Dialyze and freeze-dry for later use.

[0127] (7) After the above composition is uniformly mixed, a reaction is initiated under light conditions to obtain a product having a wavelength of 405 nm.

[0128] Except for the gelation temperature, the raw materials and preparation conditions of this comparative example are the same as those in the literature “Novel Photo-and Thermo-Responsive Nanocomposite Hydrogels Based on Functionalized rGO and ModifiedSIS / Chitosan Polymers for Localized Treatment of Malignant CutaneousMelanoma, doi:10.3389 / fbioe.2022.947616”.

[0129] The technical solution of the present invention is further illustrated by experiments below.

[0130] Experimental Example 1 Optimal ratio of small intestinal submucosal layer to methacrylic anhydride

[0131] (1) Experimental methods

[0132] The grafting rate of the modified small intestinal submucosa is closely related to the mechanical properties of the hydrogel. In this experimental example, the grafting rate is used as an indicator to optimize the feed ratio of the small intestinal submucosa to methacrylic anhydride during the preparation of the modified small intestinal submucosa.

[0133] The preferred preparation method of the modified small intestinal submucosa in this experimental example is consistent with that in Example 1, except that the mass molar ratio of free amino group to MA is set to 1:5, 1:20 and 1:100 respectively. The grafting rate of the prepared modified small intestinal submucosa material is tested by the o-phthalaldehyde (OPA) method.

[0134] (2) Experimental results

[0135] The results are as follows Figure 1 As shown, when the molar ratio of free amino group to MA is 1:20 to 1:100, the grafting rate is about 70-80%, and there is no further increase as the ratio continues to rise.

[0136] Experimental Example 2 Mechanical Properties Evaluation

[0137] (1) Experimental methods

[0138] During the application process, the small intestinal submucosal hydrogel has problems such as unstable mechanical strength. In order to verify whether the modification can improve the mechanical properties of the small intestinal submucosal hydrogel, SISMA hydrogel was prepared according to Example 5, SIS hydrogel was prepared according to Comparative Example 1, and GelMA hydrogel was prepared according to Comparative Example 2. The three hydrogels were used for rheological tests.

[0139] (2) Experimental results

[0140] The results are as follows Figure 2As shown in the figure, the rheological properties of the modified small intestinal submucosal hydrogel (SISMA) are significantly improved compared with SIS hydrogel, and the strength is higher than that of GelMA hydrogel under the same concentration conditions. This shows that the modified small intestinal submucosal hydrogel provided by the present invention has better mechanical properties.

[0141] Experimental Example 3: Determination of cytokine content

[0142] (1) Experimental methods

[0143] SISMA hydrogel was prepared according to Example 5, SIS hydrogel was prepared according to Comparative Example 1, and GelMA hydrogel was prepared according to Comparative Example 2. After SISMA, SIS and GelMA were gelled, they were centrifuged and the supernatant was taken. The ELISA experiment was used to determine the content of cytokines (VEGF, FGF-2 and TGF-β) in the supernatant. At the same time, the BCA method (Ya enzyme BCA protein quantification kit, ZJ102) was used to determine the protein content in the supernatant for standardization of factor concentration. ELISA kits all use Zhuocai biological products, including: Porcine VEGFELISA Kit (porcine vascular endothelial growth factor ELISA kit) ZC-39900, Porcine TGF-βELISA Kit (porcine transforming growth factor β ELISA kit) ZC-50020, Porcine FGF-2ELISA Kit (porcine basic fibroblast growth factor ELISA kit) ZC-39480.

[0144] (2) Experimental results

[0145] The results are as follows Figure 3 As shown, the cytokine content of the modified small intestinal submucosal hydrogel (SISMA) was not significantly changed compared with that before modification (SIS), and was significantly higher than that of the GelMA hydrogel with the same concentration. This indicates that the modified small intestinal submucosal hydrogel of the present invention has the same biological activity as that before modification.

[0146] Example 4 Gel Forming Performance

[0147] (1) Experimental methods

[0148] SISMA hydrogels were prepared according to Example 5 and Comparative Example 3, and the gelling properties of SISMA obtained by the two methods were compared.

[0149] (2) Experimental results

[0150] The results are as follows Figure 4 As shown, the gelling performance of SISMA obtained by the method of Example 5 is significantly better than that of the SISMA material prepared by Comparative 3, which is still in a fluid state and cannot form a hydrogel.

[0151] Through the above comparative experiments, it can be seen that the SISMA hydrogel preparation method provided by the present invention makes it easier for SISMA materials to form hydrogels at room temperature. In addition, the SISMA hydrogel preparation method of the present invention has the following advantages: 1) it can react at room temperature or in an ice bath, which can better retain the bioactive components; 2) it does not involve EDC / NHS, organic solvents and other components, and the reaction is greener and non-toxic.

[0152] In summary, the present invention provides a modified small intestinal submucosa, which is a methacrylic anhydride grafted small intestinal submucosa, and the methacrylic anhydride grafting rate is 40 to 90%. After cross-linking and curing, the modified small intestinal submucosa material provided by the present invention has excellent biocompatibility, degradability and mechanical properties, can effectively promote angiogenesis, regulate immune response, and has good biological activity. In addition, the present invention improves the preparation method of the modified small intestinal submucosa hydrogel, which has the advantages of mild conditions, non-toxicity, and better product gelling properties. Therefore, the modified small intestinal submucosa provided by the present invention and the method for making a hydrogel therefrom have broad application prospects.

Claims

1. A modified small intestinal submucosal material, It is characterized in that It is a small intestinal submucosa grafted with methacrylic anhydride, and the methacrylic anhydride grafting rate is 40 to 90%.

2. The modified small intestinal submucosa material according to claim 1, It is characterized in that The methacrylic anhydride grafting rate is 40 to 90%.

3. The modified small intestinal submucosa material according to claim 2, It is characterized in that The methacrylic anhydride grafting rate is 80%.

4. The modified small intestinal submucosa material according to any one of claims 1 to 3, It is characterized in that It is prepared by reacting the small intestinal submucosal layer with methacrylic anhydride in an alkaline aqueous solution.

5. The modified small intestinal submucosa material according to claim 4, It is characterized in that The molar ratio of the free amino groups in the small intestinal submucosal layer to methacrylic anhydride is 1:(5-100).

6. The modified small intestinal submucosa material according to claim 5, It is characterized in that The molar ratio of the free amino groups in the small intestinal submucosal layer to methacrylic anhydride is 1:

20.

7. The modified small intestinal submucosa material according to claim 4, It is characterized in that The alkaline aqueous solution is an aqueous solution with a pH of 8-10.

8. The modified small intestinal submucosa material according to claim 4, It is characterized in that The reaction conditions are: 0-50°C for 1-24h.

9. The modified small intestinal submucosa material according to claim 8, It is characterized in that The reaction conditions are: 0-20° C. for 20-24 hours, 20-35° C. for 10-20 hours, and 35-50° C. for 1-10 hours.

10. The modified small intestinal submucosa material according to claim 4, It is characterized in that The amino content of the small intestinal submucosal layer is 400-800 μM.

11. The modified small intestinal submucosa material according to claim 10, It is characterized in that The small intestinal submucosa is the small intestinal submucosa that has been defatted, decellularized and digested.

12. A method for preparing the modified small intestinal submucosal material according to any one of claims 1 to 11, It is characterized in that The steps include: (1) The submucosa of the small intestine dissolves in water at 35 to 50°C; (2) adding methacrylic anhydride, adjusting the pH to 8-10, and reacting at 35-50° C. for 1-10 hours; Or, comprising the steps of: (i) The small intestinal submucosa dissolves in water at 20-35°C; (ii) adding methacrylic anhydride, adjusting the pH to 8-10, and reacting at 25° C. for 10-20 hours; Or, comprising the steps of: (a) The small intestinal submucosa is dissolved in water at 0 to 4°C; (b) adding methacrylic anhydride, adjusting the pH to 8-10, and reacting at 0-20°C for 20-24 hours.

13. The preparation method according to claim 12, It is characterized in that The method also includes the following steps: adding 4 to 6 times the volume of water to terminate the reaction, dialysis, and freeze-drying.

14. A composition, It is characterized in that It comprises the modified small intestinal submucosa as described in any one of claims 1 to 11 and an initiator, wherein the weight ratio of the modified small intestinal submucosa to the initiator is 10-200:

1.

15. The composition of claim 14, It is characterized in that The weight ratio of the modified small intestinal submucosal layer to the initiator is 20-100:

1.

16. The composition of claim 14, It is characterized in that The initiator is a photoinitiator.

17. The composition of claim 16, It is characterized in that The photoinitiator is selected from at least one of LAP and I2959.

18. The composition according to any one of claims 14 to 17, It is characterized in that It also includes solvents.

19. The composition of claim 18, It is characterized in that The solvent is water.

20. Use of the composition according to any one of claims 14 to 19 for preparing a gel.

21. A gel, It is characterized in that The composition is prepared by reacting the composition described in any one of claims 14 to 19 under light conditions.

22. The gel according to claim 21, It is characterized in that The wavelength of the illumination condition is 365-405 nm.

23. The gel according to claim 22, It is characterized in that In the composition, the initiator includes LAP, and the wavelength of the illumination condition is 405 nm; Or, in the composition, the initiator includes I2959, and the wavelength of the illumination condition is 365 nm.

24. A tissue engineering scaffold, It is characterized in that The gel is obtained by freeze-drying the gel described in any one of claims 21 to 23.

25. Use of the gel according to any one of claims 21 to 23 or the tissue engineering scaffold according to claim 24 as a tissue repair material.

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