Uterus acellular matrix collagen material and preparation method thereof

Abstract

The invention discloses a uterus acellular matrix collagen material and a preparation method thereof. According to the method, the integrity of the tissue matrix structure is protected to the greatest extent while the decellularization effect is ensured in a manner of combining a natural detergent and nano silicon dioxide treatment, and the damage to the tissue matrix caused by using a chemical detergent in a traditional method is avoided. By optimizing treatment conditions including temperature, stirring rotating speed and treatment time, the method can efficiently remove cell components, meanwhile, a good tissue structure is reserved, and good biocompatibility is achieved. The material can be widely applied to tissue repair, regenerative medicine and clinical treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and specifically relates to a uterine acellular matrix gelatinous raw material and a preparation method thereof. Background Art

[0002] At present, as a natural tissue engineering material, uterine acellular matrix has been widely used in the field of tissue repair and regeneration. However, there are still certain challenges in the aspects of cell removal efficiency, tissue matrix structure protection, and biocompatibility of existing acellular matrix materials. For example, detergents (SDS, Triton-X100) in traditional acellular methods may damage the matrix structure, affect the biocompatibility of the material, and the acellular effect is uneven, which limits its clinical application. Therefore, there is an urgent need for a new method that can improve the acellular efficiency and ensure the biocompatibility of the material while maintaining a good tissue matrix structure. Summary of the Invention

[0003] The purpose of the present invention is to provide a uterine acellular matrix gelatinous raw material and a preparation method thereof, which have the advantages of high-efficiency acellularization, good protection of tissue matrix structure, and excellent biocompatibility, and solve the problems of low acellular efficiency and poor biocompatibility in the prior art.

[0004] To achieve the above purpose, the present invention provides the following technical solution: A preparation method of a uterine acellular matrix gelatinous raw material, comprising the following steps:

[0005] S1. Tissue pretreatment;

[0006] S2. Nano-silica treatment;

[0007] S3. Detergent treatment;

[0008] S4. Enzymatic hydrolysis treatment;

[0009] S5. Rinsing;

[0010] S6. Non-ionic detergent washing;

[0011] S7. Nuclease treatment;

[0012] S8. Rinsing;

[0013] S9. Punching and disinfection;

[0014] S10. Freeze-drying.

[0015] Preferably, in the S1, it further includes:

[0016] S11. Take fresh porcine uterine tissue, cut it along the midline, and mechanically remove the serosa layer and muscular layer with a surgical blade, only retaining the endometrium layer and the subendometrial stroma layer;

[0017] S12. Rinse the tissue after removing the serosa and muscular layer with normal saline to remove residual blood and tissue debris on the surface;

[0018] The following steps are further included in S2:

[0019] S21. Place the processed tissue in a nano-silica solution, where the concentration of nano-silica particles is 0.1 mg / mL to 1 mg / mL and its surface is modified with PEG;

[0020] S22. Process at 4°C and 100 rpm for 48 hours to ensure that the nano-silica fully penetrates into the tissue.

[0021] Preferably, the following steps are further included in S3:

[0022] S31. Transfer the tissue in step S2 to a solution containing a natural detergent, where the natural detergent is oleanolic acid saponin or cocoate, and the concentration is 0.1 - 1% (w / v);

[0023] S32. Stir at 4°C and 130 rpm for 12 hours to ensure that the detergent fully acts on the tissue;

[0024] The following steps are further included in S4:

[0025] S41. Place the tissue in step S3 in a trypsin or pancreatic lipase solution, with a concentration of 0.1 - 1% (w / v), control the temperature at 37°C, and the stirring speed at 100 - 150 rpm for 12 hours;

[0026] S42. Rinse the enzymatically digested tissue with PBS to remove the residual enzyme.

[0027] Preferably, the following steps are further included in S5:

[0028] S51. Take out the tissue in step S4 and repeatedly rinse it with PBS and deionized water respectively for 30 minutes;

[0029] S52. Change the liquid every 15 minutes to ensure complete removal of the residual reagent;

[0030] The following steps are further included in S6:

[0031] S61. Place the rinsed tissue in a 10 mM Tris-HCl solution containing 0.1% Triton X-100, at a temperature of 4°C, with a stirring speed of 130 - 150 rpm, and the cleaning time is 48 hours;

[0032] S62. Change the liquid every 12 hours to ensure complete removal of the residual detergent.

[0033] Preferably, S7 further includes:

[0034] S71. Take out the washed tissue, place it in a nuclease solution, stir at 37°C - 40°C for 12 - 15 hours to remove residual nucleic acid components;

[0035] S72. Rinse the tissue treated with nuclease with PBS to remove the residual nuclease;

[0036] Preferably, S8 further includes:

[0037] S81. Take out the tissue treated with nuclease and rinse it repeatedly again to ensure that all residual reagents are removed;

[0038] S82. Change the liquid every 15 minutes to ensure complete removal of residual reagents.

[0039] Preferably, S9 further includes:

[0040] S91. Place the rinsed tissue in a 5% peracetic acid solution, stir at 80 - 100 rpm at 4°C for 2 - 3 hours;

[0041] S92. Rinse the tissue after punching treatment with deionized water to remove the residual peracetic acid;

[0042] Preferably, S10 further includes:

[0043] S101. Spread the treated tissue flat on a sterilized plastic plate and place it in a freeze dryer for freeze-drying;

[0044] S102. Store the freeze-dried tissue at -20°C.

[0045] Preferably, in S41, the concentration of the trypsin or lipase is 0.5% (w / v), and the stirring speed is 120 rpm.

[0046] Preferably, in S61, the cleaning time is 48 hours, and the stirring speed is 130 rpm.

[0047] Preferably, in S71, the nuclease treatment time is 12 hours, and the temperature is 37°C;

[0048] In S91, the punching treatment time is 2 hours, and the stirring speed is 100 rpm.

[0049] A raw material for uterine acellular matrix glue, wherein the glue raw material is prepared by the method according to any one of claims 1 - 9.

[0050] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0051] 1. The present invention replaces traditional chemical detergents with natural detergents (oleanolic acid saponins, cocoate), effectively avoiding damage to the matrix structure while maintaining the biocompatibility of the material.

[0052] 2. The combination of nano-silica and surface PEG modification is adopted to improve the uniformity and efficiency of decellularization.

[0053] 3. Enzymatic treatment and nuclease treatment during the preparation process further improve the decellularization efficiency and ensure the complete removal of cellular components.

[0054] 4. The final product has good biodegradability and biocompatibility and is suitable for clinical tissue repair. Description of the Drawings

[0055] Figure 1 It is a flowchart of the preparation method of a uterine acellular matrix gelatin material of the present invention.

[0056] Figure 2 It is an experimental photo of the preparation process and results of a porcine uterine acellular matrix gelatin material of the present invention. Detailed Embodiments

[0057] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0058] A preparation method of a uterine acellular matrix gelatin material:

[0059] Tissue Pretreatment (S1)

[0060] S11: Take fresh porcine uterine tissue, cut it open along the midline, and mechanically remove the serosa layer and muscular layer with a surgical blade, leaving only the endometrium layer and the subendometrial stroma layer. The purpose of this step is to reduce the tissue layers, lower the decellularization difficulty, and improve the decellularization efficiency.

[0061] S12: Rinse the tissue after removing the serosa layer and muscular layer with physiological saline to remove the residual blood and tissue fragments on the surface. This step ensures the cleanliness of the tissue and lays a foundation for subsequent processing.

[0062] Nano-silica Treatment (S2)

[0063] S21: Place the processed tissue in a nano-silica solution. The concentration of the nano-silica particles is 0.1 mg / mL to 1 mg / mL, and its surface is modified with PEG. The use of nano-silica helps to improve the decellularization efficiency, and the PEG modification increases the biocompatibility of the nano-silica.

[0064] S22: Process at 4°C and 100 rpm for 48 hours to ensure that the nano-silica fully penetrates into the tissue. The purpose of this step is to further improve the decellularization effect by utilizing the properties of the nano-silica.

[0065] Detergent treatment (S3)

[0066] S31: Transfer the tissue from step S2 to a solution containing a natural detergent. The natural detergent is oleanolic acid saponin or caprylate, and the concentration is 0.1 - 1% (w / v). The use of the natural detergent avoids the damage to the tissue structure caused by traditional chemical detergents.

[0067] S32: Stir at 4°C and 130 rpm for 12 hours to ensure that the detergent fully acts on the tissue. The purpose of this step is to further remove the cellular components in the tissue by utilizing the properties of the detergent.

[0068] Enzymatic digestion treatment (S4)

[0069] S41: Place the tissue from step S3 in a trypsin or pancreatic lipase solution with a concentration of 0.1 - 1% (w / v), control the temperature at 37°C, and the stirring speed at 100 - 150 rpm for 12 hours. The enzymatic digestion treatment helps to completely remove the cellular components in the tissue.

[0070] S42: Rinse the enzymatically digested tissue with PBS to remove the residual enzymes. This step ensures that there are no residual enzymes in the tissue and avoids affecting subsequent treatments.

[0071] Rinsing (S5)

[0072] S51: Take out the tissue from step S4 and repeatedly rinse it with PBS and deionized water for 30 minutes. The purpose of this step is to remove the residual chemical reagents in the tissue.

[0073] S52: Change the liquid every 15 minutes to ensure complete removal of the residual reagents. This step ensures the cleanliness of the tissue and lays the foundation for subsequent treatments.

[0074] Non-ionic detergent washing (S6)

[0075] S61: Place the rinsed tissue in a 10 mM Tris-HCl solution containing 0.1% Triton X-100 at 4°C with a stirring speed of 130 - 150 rpm for 48 hours. The use of the non-ionic detergent helps to further remove cellular components in the tissue.

[0076] S62: Change the solution every 12 hours to ensure complete removal of residual detergent. This step ensures that there is no residual detergent in the tissue, avoiding interference with subsequent processing.

[0077] Nuclease treatment (S7)

[0078] S71: Take out the rinsed tissue, place it in a nuclease solution, and stir at 37°C - 40°C for 12 - 15 hours to remove residual nucleic acid components. Nuclease treatment helps to completely remove nucleic acid components in the tissue, avoiding immune reactions.

[0079] S72: Rinse the tissue treated with nuclease with PBS to remove residual nuclease. This step ensures that there is no residual nuclease in the tissue, avoiding interference with subsequent processing.

[0080] Rinsing (S8)

[0081] S81: Take out the tissue treated with nuclease and rinse it repeatedly to ensure removal of all residual reagents. The purpose of this step is to ensure that there are no residual chemical reagents in the tissue.

[0082] S82: Change the solution every 15 minutes to ensure complete removal of residual reagents. This step ensures the cleanliness of the tissue, laying the foundation for subsequent processing.

[0083] Punching and disinfection (S9)

[0084] S91: Place the rinsed tissue in a 5% peracetic acid solution and stir at 80 - 100 rpm at 4°C for 2 - 3 hours. Punching treatment helps to increase the permeability of the tissue, facilitating subsequent processing.

[0085] S92: Rinse the tissue treated by punching with deionized water to remove residual peracetic acid. This step ensures that there is no residual peracetic acid in the tissue, avoiding interference with subsequent processing.

[0086] Freeze-drying (S10)

[0087] S101: Place the treated tissue flat on a sterilized plastic plate and perform freeze-drying in a freeze-dryer. Freeze-drying treatment helps to remove water in the tissue and extend the storage time.

[0088] S102: The freeze-dried tissue is stored at -20°C. This step ensures the stability and long-term preservation of the tissue.

[0089] Figure 2 Description:

[0090] Figure A: Appearance photo of fresh porcine uterine tissue, showing the original state of the tissue.

[0091] Figure B: Photo of the tissue after being cut along the midline and removing the serosa layer and muscular layer, only retaining the endometrial layer and the subendometrial stromal layer.

[0092] Figure C: Photo of the tissue after treatment with nano-silica, showing the distribution of nano-silica in the tissue.

[0093] Figure D: Photo of the tissue after treatment with detergent, showing the tissue structure after detergent treatment.

[0094] Figure E: Microscopic image of the tissue after enzymatic digestion, showing the removal of cell components.

[0095] Figure F: Microscopic image of the tissue after washing with non-ionic detergent, showing the tissue structure after further removal of cell components.

[0096] Figure G: Microscopic image of the tissue after treatment with nuclease, showing the removal of nucleic acid components.

[0097] Example 1: Application of uterine acellular matrix gelatinous raw material in tissue engineering

[0098] In the field of tissue engineering, uterine acellular matrix gelatinous raw material, as a natural extracellular matrix scaffold, has good biocompatibility and biodegradability, and can provide an ideal microenvironment for cell attachment, proliferation and differentiation. However, traditional decellularization methods often lead to damage to the tissue matrix structure, affecting its biocompatibility and mechanical properties.

[0099] This example aims to prepare a uterine acellular matrix gelatinous raw material with high decellularization efficiency, good protection of tissue matrix structure and excellent biocompatibility through the preparation method of the present invention for cell culture and tissue repair in tissue engineering.

[0100] Using natural detergents (oleanolic acid saponin, coconut oilate) to replace traditional chemical detergents avoids damage to the tissue matrix structure.

[0101] Adopting the combination of nano-silica and surface PEG modification improves the uniformity and efficiency of decellularization.

[0102] By optimizing the treatment conditions, including temperature, stirring speed and treatment time, thorough removal of cell components is ensured while retaining a good tissue structure.

[0103] Application scenarios: The uterine acellular matrix gel raw material of this embodiment can be widely applied to cell culture in tissue engineering, tissue repair, and the field of regenerative medicine, such as endometrial repair, scar tissue repair, etc.

[0104] Comparative experiment:

[0105] Performance indicators The method of the present invention The prior art method Decellularization efficiency 95％ 80％ Integrity of tissue matrix structure 90％ 70％ Biocompatibility 95％ 85％ Mechanical properties (tensile strength) 50MPa 30MPa Preparation period 8 days 12 days Preparation cost 500 yuan 1000 yuan

[0106] Example 2: Application of uterine acellular matrix gel raw material in clinical treatment

[0107] In clinical treatment, the uterine acellular matrix gel raw material can be used to treat diseases such as uterine infertility and endometrial injury. Traditional treatment methods often require multiple surgeries and have unsatisfactory effects. The preparation method of the present invention can provide a more effective treatment material and improve the treatment effect.

[0108] This embodiment aims to prepare a uterine acellular matrix gel raw material with high decellularization efficiency, good tissue matrix structure protection, and excellent biocompatibility through the preparation method of the present invention for endometrial repair and tissue regeneration in clinical treatment.

[0109] Using natural detergents (oleic acid saponin, coconut oilate) to replace traditional chemical detergents avoids damage to the tissue matrix structure.

[0110] Adopting the combination of nano-silica and surface PEG modification improves the uniformity and efficiency of decellularization.

[0111] By optimizing the treatment conditions, including temperature, stirring speed, and treatment time, it is ensured that the cell components are completely removed while retaining a good tissue structure.

[0112] The uterine acellular matrix gel raw material of this embodiment can be widely applied to endometrial repair, scar tissue repair, etc. in clinical treatment, improving the treatment effect and reducing the pain of patients.

[0113] Comparative experiment:

[0114]

[0115] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a uterine acellular matrix collagen material, characterized in that: The following steps are involved: S1, tissue pretreatment; S2, nano-silica treatment; S3, detergent treatment; S4, enzymatic treatment; S5, rinsing; S6, nonionic detergent washing; S7, nuclease treatment; S8, rinsing; S9, drilling and disinfection; S10. Freeze-drying.

2. The method for preparing a uterine acellular matrix collagen material according to claim 1, characterized in that: Said S1 further comprises: S11. Take fresh porcine uterine tissue, cut it open along the midline, and use a surgical blade to mechanically remove the serosa and muscular layer, leaving only the endometrium and subendometrial matrix layer; S12, rinse the tissue after removing the serosal layer and muscle layer with normal saline to remove the blood and tissue fragments remaining on the surface; Said S2 further comprises: S21, placing the treated tissue in a nano-silica solution, wherein the nano-silica particle concentration is 0.1 mg / mL to 1 mg / mL, and the surface of the nano-silica particle is modified with PEG; S22. Treat at 4°C and 100 rpm for 48 hours to ensure that the nano-silica fully penetrates into the tissue.

3. The method for preparing a uterine acellular matrix collagen material according to claim 1, characterized in that: The S3 further comprises: S31, transferring the tissue in step S2 into a solution containing a natural detergent, wherein the natural detergent is olive oil saponin or coconut oil salt, with a concentration of 0.1-1% (w / v); S32, agitate at 4°C, 130 rpm for 12 hours to ensure that the detergent is fully applied to the tissue; The S4 further comprises: S41, placing the tissue in step S3 in a trypsin or pancreatic lipase solution at a concentration of 0.1-1% (w / v), the temperature is controlled at 37° C., the stirring speed is 100-150 rpm, and the duration is 12 hours; S42. Rinse the tissue after enzymatic hydrolysis with PBS to remove residual enzyme.

4. The method for preparing a uterine acellular matrix collagen material according to claim 1, characterized in that: The S5 further comprises: S51, taking out the tissue in step S4, and repeatedly rinsing it with PBS and deionized water respectively, the rinsing time is 30 minutes; S52, change the solution every 15 minutes to ensure that residual reagents are completely removed; The S6 further comprises: S61, placing the rinsed tissue in a 10 mM Tris-HCl solution containing 0.1% Triton X-100 at a temperature of 4°C and a stirring speed of 130-150 rpm for 48 hours; S62. Change the solution every 12 hours to ensure that residual detergent is completely removed.

5. The method for preparing a uterine acellular matrix collagen material according to claim 1, characterized in that: The S7 further comprises: S71. Take out the cleaned tissue, place it in a nuclease solution, and stir it at 37°C-40°C for 12-15 hours to remove residual nucleic acid components; S72, rinsing the nuclease-treated tissue with PBS to remove residual nuclease; The S8 further comprises: S81, remove the tissue after nuclease treatment, and rinse it repeatedly again to ensure that all residual reagents are removed; S82. Change the solution every 15 minutes to ensure that residual reagents are completely removed.

6. The method for preparing a uterine acellular matrix collagen material according to claim 1, characterized in that: The S9 further comprises: S91, placing the rinsed tissue in a 5% peracetic acid solution, stirring at 80-100 rpm and 4°C for 2-3 hours; S92, rinsing the punched tissue with deionized water to remove residual peracetic acid; The S10 further includes: S101, spreading the treated tissue on a sterilized plastic plate, and placing it in a freeze dryer for freeze drying; S102. The freeze-dried tissue was stored at -20°C.

7. The method for preparing a uterine acellular matrix collagen material according to claim 1, characterized in that: In S41, the concentration of trypsin or pancreatic lipase is 0.5% (w / v), and the stirring speed is 120 rpm.

8. The method for preparing a uterine acellular matrix collagen material according to claim 1, characterized in that: In S61, the cleaning time is 48 hours and the stirring speed is 130 rpm.

9. The method for preparing a uterine acellular matrix collagen material according to claim 1, characterized in that: In S71, the nuclease treatment time is 12 hours at a temperature of 37°C; In S91, the punching treatment time is 2 hours and the stirring speed is 100 rpm.

10. A uterine decellularized matrix collagen material, characterized in that: The collagen material is prepared by the method according to any one of claims 1-9.

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