Fat acellular matrix self-crosslinking hydrogel as well as preparation method and application thereof

Abstract

The invention relates to fat acellular matrix self-crosslinking hydrogel as well as a preparation method and application thereof. According to the preparation method, pretreatment is performed by applying a physical method, and deep degreasing and decellularization treatment is performed on adipose tissues by properly using chemical and biological methods, so that cell components are removed to the greatest extent, a pure extracellular matrix structure is reserved, the preparation efficiency of the extracellular matrix is improved, and the preparation cost is reduced. And the contact between the material and chemical and biological reagents is reduced. And the mesoporous molecular sieve is added in the preparation method to perform physical adsorption on the biological enzyme, so that the operation difficulty is simplified, and large-scale production is facilitated. The pepsin is removed by the mesoporous molecular sieve, so that the residue of biological enzyme is reduced as much as possible, and the applicable range of the extracellular matrix hydrogel is widened.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and in particular to a fat acellular matrix self-crosslinking hydrogel and a preparation method and application thereof. Background Art

[0002] In today's society, people's pursuit of beauty is constantly improving, and the demand for facial rejuvenation and beauty is becoming stronger. Problems such as facial depressions and wrinkles not only affect one's appearance but can also have a negative psychological impact, reducing self-confidence.

[0003] With the accelerated pace of life and people's increasing acceptance of minimally invasive surgery, injectable facial fillers have attracted widespread attention due to their advantages such as ease of operation, minimal trauma, and quick recovery. However, existing facial fillers have many shortcomings, such as: (1) Although traditional solid implant materials such as silicone and expanded polytetrafluoroethylene can improve facial contours to a certain extent, the surgical risks are relatively high and may cause complications such as infection, displacement, and rejection. In addition, the appearance and touch of these materials may not be natural enough, affecting the aesthetics of the face.

[0004] (2) Autologous fat transplantation has problems such as donor site damage, high fat absorption rate, and the need for multiple surgeries. During the operation, fat needs to be extracted from other parts of the patient's body, which will cause additional pain and trauma to the patient. Moreover, the survival rate of transplanted fat is unstable, and some fat may be absorbed, requiring multiple surgeries to achieve the ideal filling effect.

[0005] (3) Injectable synthetic polymer materials such as hyaluronic acid have a short lifespan, usually only lasting a few months to a year, and require repeated injections. Frequent injections not only increase the patient's pain and financial burden, but may also cause adverse reactions such as allergic reactions and local swelling.

[0006] (4) Animal-derived collagen can be treated to reduce its immunogenicity, but it still has the potential to trigger an immune response. Different individuals have different degrees of immune response to animal collagen, which may cause adverse reactions such as local redness, itching, and pain, and may even cause severe allergic reactions, affecting the effectiveness and safety of facial fillers. Animal-derived materials also have the potential risk of transmitting animal-derived diseases.

[0007] Therefore, finding safe and effective facial filling materials has become a common goal for many beauty seekers and medical workers. Summary of the Invention

[0008] The purpose of the present invention is to disclose a self-crosslinked adipose decellularized matrix hydrogel and its preparation method and application, so as to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0009] The first aspect of the present invention is to provide a method for preparing adipose acellular matrix self-crosslinking hydrogel.

[0010] The second aspect of the present invention is to provide a self-crosslinked adipose decellularized matrix hydrogel obtained by the preparation method described in the first aspect of the present invention.

[0011] The third aspect of the present invention is to provide a use of the adipose decellularized matrix self-crosslinking hydrogel according to the second aspect of the present invention.

[0012] The preparation method according to the first aspect of the present invention comprises the following steps: S1. Collect human adipose tissue, wash it at least three times with cleaning solution, centrifuge and discard the upper fat layer and lower liquid layer, and collect only the middle adipose tissue layer; S2, mechanically disrupting the adipose tissue layer, adding sterile distilled water for centrifugation, discarding the upper layer of fat, retaining the lower layer of tissue, and then adding a cleaning solution for ultrasonic cleaning; S3. Add degreasing reagent to the cleaned tissue layer and shake it, then centrifuge it and retain the precipitate; S4, replacing the delipidation reagent with a decellularization reagent, and repeating step S3; S5. The decellularized precipitate is freeze-dried and sterilized to obtain a sterile adipose tissue extracellular matrix; S6, grinding the sterile adipose tissue extracellular matrix, adding a digestion reagent, mixing, and shaking to digest until no particles are visible to the naked eye, to obtain a sol; S7, adding mesoporous molecular sieves to the sol, shaking to absorb excess digestion reagent, and centrifuging to remove precipitates to obtain extracellular matrix; S8. Use a neutralizing reagent pre-frozen at 4°C to adjust the pH value of the extracellular matrix to 6.8-7.5, and add a buffer reagent pre-frozen at 4°C to adjust the buffer salt concentration to obtain a sterile injectable pregel, which is stored at 4°C.

[0013] The preparation method uses human adipose tissue as the primary material and uses a combination of physical, chemical, and biological methods to perform fat decellularization, achieving deep degreasing and decellularization, while preserving the extracellular matrix structure to the greatest extent possible. Furthermore, the preparation method abandons the traditional dialysis purification process and instead uses mesoporous molecular sieves to efficiently remove residual digestion reagents, significantly reducing the time cost of the preparation method, simplifying the operational difficulty, and achieving the beneficial effect of reducing production costs. The resulting material is also temperature-sensitive, maintaining fluidity at low temperatures and capable of self-crosslinking and solidification at body temperature.

[0014] In a further application embodiment, the centrifugation operating parameters of step S1 are 2000-4000 g, and the centrifugation time is 5-8 minutes.

[0015] In a further application embodiment, the centrifugal operating parameters in step S2 are 3000-6000 g, and the centrifugation time is 6-10 minutes.

[0016] In a further application embodiment, the centrifugation operating parameters of steps S3 and S4 are 7000-10000 g, and the centrifugation time is 8-10 minutes.

[0017] In a further embodiment, the centrifugation operating parameters in step S7 are 3000-5000 g, and the centrifugation time is 8-10 minutes.

[0018] In a further application embodiment, the cleaning solution is selected from one of sterile distilled water, sterile physiological saline or sterile phosphate buffered saline (PBS).

[0019] In a further application embodiment, the decellularization reagents include decellularization reagent A, decellularization reagent B, decellularization reagent C and decellularization reagent D. The precipitate needs to be shaken and centrifuged with the four decellularization reagents in sequence to remove the eluted cells.

[0020] In a further application embodiment, the decellularization reagent A is selected from a sodium chloride (NaCl) solution with a concentration of 0.8-1.2 mol / L, a hydrochloric acid (HCl) solution with a concentration of 0.05-0.12 mol / L, or a sodium hydroxide (NaOH) solution with a concentration of 0.1-0.15 mol / L.

[0021] In a further application embodiment, the decellularization reagent B is selected from a sodium dodecyl sulfate (SDS) solution with a volume fraction of 0.5-1% or a sodium deoxycholate (SDC) solution with a volume fraction of 2-4%.

[0022] In a further application embodiment, the decellularization reagent C is selected from one of a Triton X-100 solution with a volume fraction of 0.5-1% or a 3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt (CHAPS) solution with a concentration of 6-10 mmol / L.

[0023] In a further application embodiment, the decellularization reagent D is selected from a trypsin solution with a volume fraction of 0.03-0.08% or a pepsin solution with a concentration of 0.5-1.5 mg / mL.

[0024] In a further application embodiment, the ratio of the tissue layer to the degreasing reagent in step S3 is 1 g: (8-15) mL.

[0025] In a further application embodiment, the ratio of the precipitate to the decellularization reagent in step S4 is 1 g: (8-15) mL.

[0026] In a further application embodiment, the operating parameters of the ultrasonic cleaning treatment are 20-100 kHz, and the cleaning time is 1-2 hours.

[0027] In a further application embodiment, the degreasing agent is selected from one of methanol, isopropanol or anhydrous ethanol.

[0028] In a further application embodiment, the freeze-drying processing parameters are vacuuming to below 50 Pa, temperature -40~-60°C, and time of 10~20 hours.

[0029] In a further application embodiment, the digestion reagent includes digestion reagent A and digestion reagent B, and the sterile adipose tissue extracellular matrix and the two digestion reagents need to be shaken and centrifuged in sequence to remove the decomposed impurities.

[0030] In a further application embodiment, the digestion reagent A is selected from a HCl solution with a concentration of 0.01-0.02 mol / L or an acetic acid (CH3COOH) solution with a concentration of 0.1-0.5 mol / L.

[0031] In a further application embodiment, the digestion reagent B is selected from trypsin with a concentration of 2-5 mg / mL or pepsin with a concentration of 0.8-1.5 mg / mL.

[0032] In further embodiments, the mesoporous molecular sieve is selected from MCM-41, MCM-48, MCM-50, SBA-15, SBA-11, SBA-16, SBA-2, HMS, or KIT-6. Preferably, the mesoporous molecular sieve is SBA-15 or MCM-41. The amount of the mesoporous molecular sieve used is 2 to 10 times that of the digestion reagent B.

[0033] The adipose decellularized matrix self-crosslinked hydrogel described in the second aspect of the present invention is prepared by the above-mentioned preparation method. It has the following advantages: Good biocompatibility: Adipose decellularized matrix retains the three-dimensional structure and bioactive components of natural tissue, has good compatibility with human tissue, and can promote cell adhesion, growth and differentiation.

[0034] Degradability: Adipose decellularized matrix can be gradually degraded and absorbed in the body and will not remain for a long time, thus avoiding the occurrence of foreign body reaction.

[0035] Low immunogenicity: After removing the cellular components, the immunogenicity of the adipose decellularized matrix is ​​greatly reduced, reducing the risk of rejection.

[0036] Abundant sources: Adipose tissue is widely distributed in the human body and is relatively easy to obtain. It can be obtained in large quantities through methods such as liposuction, making large-scale preparation possible.

[0037] Rich in nutrients: Adipose decellularized matrix contains a large amount of collagen, elastin and fibronectin, and is also rich in many growth factors required by the human body. It can not only provide support for the sunken parts of the face, but also provide a large amount of nutrients needed for growth, stimulating the regeneration of facial depressions.

[0038] The use described in the third aspect of the present invention refers to the use of the self-crosslinked adipose matrix hydrogel as a subcutaneous filling material in medical, cosmetic and other scenarios. The self-crosslinked hydrogel can form a stable three-dimensional network structure at a specific temperature. This self-crosslinking property enables the hydrogel to solidify rapidly after being injected into the face, maintain a stable shape, and provide long-lasting support for the sunken parts of the face. And there is no need to add other chemical crosslinking agents, eliminate the possibility of chemical crosslinking agent residues, and is more patient-friendly. The hydrogel after self-crosslinking has excellent flexibility, can fit tightly with facial tissue, and deforms naturally with the movement of facial muscles without producing stiffness, ensuring natural and smooth facial expressions. Patients do not need to go through the pain and long recovery period of traditional surgery. It can be completed by injection through minimally invasive methods such as syringes. It is simple to operate, has little trauma, and recovers quickly, greatly improving the acceptance and convenience of facial filling. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a photograph of the adipose decellularized matrix self-crosslinked hydrogel prepared in Example 1 at low temperature (4-10°C); Figure 2 This is a photo of the adipose decellularized matrix self-crosslinked hydrogel prepared in Example 1 after self-crosslinking at 37°C. DETAILED DESCRIPTION

[0040] The following examples further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0041] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0042] Example 1 S1. Obtain human allogeneic adipose tissue by vacuum liposuction. Wash the adipose tissue at least three times with PBS until no blood or other tissue particles are visible. Centrifuge the washed adipose tissue at 3000 g for 6 minutes. Discard the upper lipid layer and lower liquid layer, and collect the middle adipose tissue layer.

[0043] S2. The collected adipose tissue layer was mechanically crushed using a tissue grinder. After weighing, sterile distilled water was added according to the mass-to-volume ratio of the crushed adipose tissue of 1:8 (g:mL). The mixture was centrifuged at a centrifugal force of 5000 g for 8 minutes. The upper layer of fat was discarded, and the lower layer of tissue was retained. PBS was added for ultrasonic cleaning at a frequency of 25 kHz for 1.5 hours to clean the obtained tissue layer.

[0044] S3. Add degreasing agent isopropanol according to the mass volume ratio of the crushed adipose tissue at 1:10 (g:mL). Place on a shaker at 150 rpm for 2 hours, then centrifuge at 8000 g for 9 minutes. Discard the degreasing agent, retain the precipitate, and wash with PBS.

[0045] S4. Add 1 mol / L NaCl solution at a mass-to-volume ratio of 1:12 (g:mL) to the precipitate. Place the precipitate on a shaker at 150 rpm for 2 hours, then centrifuge at 8000 g for 9 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0046] S5. Add 0.8% SDS solution by volume at a 1:12 (g:mL) mass-to-volume ratio of the precipitate. Place the tube on a shaker at 150 rpm for 2 hours, then centrifuge at 8000 g for 9 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0047] S6. Add 0.8% Triton X-100 solution by volume at a 1:12 (g:mL) mass-to-volume ratio of the precipitate. Place the tube on a shaker at 150 rpm for 2 hours, then centrifuge at 8000 g for 9 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0048] S7. Add 0.05% trypsin solution by volume at a 1:12 (g:mL) mass-to-volume ratio of the precipitate. Place the tube on a shaker at 150 rpm for 2 hours, then centrifuge at 8000 g for 9 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0049] S8. Place the decellularized precipitate in a vacuum freeze-drying device, evacuate to a pressure below 50 Pa, maintain the temperature at -50°C, dry for 15 hours, and then sterilize with ultraviolet light to obtain a sterile adipose tissue extracellular matrix.

[0050] S9. Grind 10 mg of sterile adipose tissue extracellular matrix into powder, add 1 mL of 0.015 mol / L HCl and 3 mg of trypsin and mix well. The final concentration of sterile adipose tissue extracellular matrix in the mixture is 10 mg / mL. Place it on a shaker at 300 rpm for 48 hours for digestion until no particles are visible to the naked eye. Obtain the digested sterile extracellular matrix sol and refrigerate it at 4°C.

[0051] S10. Add 15 mg of mesoporous molecular sieve SBA-15 to the digested sterile extracellular matrix sol, place it on a shaker at 200 rpm for 2 hours, and then centrifuge it at 5000 g for 8 minutes. Discard the precipitate and retain the extracellular matrix on the top layer.

[0052] S11. Adjust the pH value of the sterile extracellular matrix sol to 7.2 with 0.1 mol / L NaOH solution, and adjust the buffer salt concentration to 0.05 mol / L with 0.1 mol / L PBS to obtain a sterile injectable pregel, which was stored at 4°C.

[0053] The prepared adipose decellularized matrix self-crosslinked hydrogel is as follows Figure 1 As shown, it has good fluidity and can be easily extruded even with a 30G needle. Figure 2 This is a photo of the fat acellular matrix self-crosslinking hydrogel completing self-crosslinking in a body temperature (37°C) environment. It solidifies in the tube and loses fluidity, which can meet the rigidity requirements of the implant material.

[0054] Example 2 S1. Obtain allogeneic adipose tissue using hydrodynamic liposuction. Wash the adipose tissue at least three times with sterile saline until no blood or other tissue particles are visible. Centrifuge the washed adipose tissue at 3500 g for 7 minutes. Discard the upper lipid layer and lower liquid layer, and collect the middle adipose tissue layer.

[0055] S2. The collected adipose tissue layer was mechanically crushed using a tissue grinder. After weighing, sterile distilled water was added according to the mass-to-volume ratio of the crushed adipose tissue of 1:9 (g:mL). The mixture was centrifuged at a centrifugal force of 5500 g for 9 minutes. The upper layer of fat was discarded, and the lower layer of tissue was retained. PBS was added for ultrasonic cleaning at an ultrasonic frequency of 50 kHz for 2 hours to clean the obtained tissue layer.

[0056] S3. Add degreasing agent methanol according to the mass volume ratio of the crushed adipose tissue at 1:13 (g:mL). Place on a shaker at 180 rpm for 2.5 hours, then centrifuge at 9000 g for 9.5 minutes. Discard the degreasing agent, retain the precipitate, and wash with PBS.

[0057] S4. Add 1.1 mol / L NaCl solution at a mass-to-volume ratio of 1:14 (g:mL) to the precipitate. Place the solution on a shaker at 180 rpm for 2.5 hours, then centrifuge at 9000 g for 9.5 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0058] S5. Add 3% SDC solution by volume at a mass-to-volume ratio of 1:14 (g:mL) to the precipitate. Shake on a shaker at 180 rpm for 2.5 hours, then centrifuge at 9000 g for 9.5 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0059] S6. Add 0.7% Triton X-100 solution by volume at a 1:14 (g:mL) mass-to-volume ratio of the precipitate. Place the tube on a shaker at 180 rpm for 2.5 hours, then centrifuge at 9000 g for 9.5 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0060] S7. Add 0.06% trypsin solution by volume at a 1:14 (g:mL) mass-to-volume ratio of the precipitate. Place the tube on a shaker at 180 rpm for 2.5 hours, then centrifuge at 9000 g for 9.5 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0061] S8. Place the decellularized precipitate in a vacuum freeze-drying device, evacuate to a pressure below 50 Pa, maintain the temperature at -55°C, and dry for 18 hours. Then sterilize with ultraviolet light to obtain a sterile adipose tissue extracellular matrix.

[0062] S9. Grind 12 mg of sterile adipose tissue extracellular matrix into powder, add 1 mL of 0.018 mol / L HCl and 4 mg of trypsin and mix well. The final concentration of sterile adipose tissue extracellular matrix in the mixture is 12 mg / mL. Place the mixture on a shaker at 350 rpm for 60 hours for digestion until no particles are visible to the naked eye. Obtain the digested sterile extracellular matrix sol and refrigerate at 4°C.

[0063] S10. Add 15 mg of mesoporous molecular sieve SBA-15 to the digested sterile extracellular matrix sol, place it on a shaker at 300 rpm for 1.6 hours, and then centrifuge it at 3200 g for 9 minutes. Discard the precipitate and retain the extracellular matrix on the top layer.

[0064] S11. Adjust the pH value of the sterile extracellular matrix sol to 7.3 with 0.1 mol / L NaOH solution, and adjust the buffer salt concentration to 0.06 mol / L with 0.1 mol / L PBS to obtain a sterile injectable pregel, which was stored at 4°C.

[0065] Example 3 S1. Obtain allogeneic human adipose tissue by ultrasonic liposuction. Wash the adipose tissue at least three times with sterile distilled water until no blood or other tissue particles are visible. Centrifuge the washed adipose tissue at 4000 g for 8 minutes. Discard the upper lipid layer and lower liquid layer, and collect the middle adipose tissue layer.

[0066] S2. The collected adipose tissue layer was mechanically crushed using a tissue grinder. After weighing, sterile distilled water was added according to a mass-to-volume ratio of 1:7 (g:mL) after crushing. The mixture was centrifuged at a centrifugal force of 4500 g for 7 minutes. The upper layer of fat was discarded, and the lower tissue layer was retained. Sterile physiological saline was added for ultrasonic cleaning at an ultrasonic frequency of 60 kHz for 1.3 hours to clean the obtained tissue layer.

[0067] S3. Add degreasing agent anhydrous ethanol to the crushed adipose tissue at a mass-to-volume ratio of 1:11 (g:mL). Place the tissue on a shaker at 160 rpm for 2 hours, then centrifuge at 8500 g for 9 minutes. Discard the degreasing agent, retain the precipitate, and wash with PBS.

[0068] S4. Add 1.0 mol / L NaCl solution at a mass-to-volume ratio of 1:13 (g:mL) to the precipitate. Place the precipitate on a shaker at 160 rpm for 2 hours, then centrifuge at 8500 g for 9 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0069] S5. Add 2.5% SDS solution by volume at a 1:13 (g:mL) mass-to-volume ratio of the precipitate. Place the tube on a shaker at 160 rpm for 2 hours, then centrifuge at 8500 g for 9 minutes. Remove the supernatant liquid, retain the precipitate, and wash with PBS.

[0070] S6. Add 0.6% Triton X-100 solution by volume at a 1:13 (g:mL) mass-to-volume ratio of the precipitate. Shake the tube at 160 rpm for 2 h, then centrifuge at 8500 g for 9 min. Remove the supernatant, retain the precipitate, and wash with PBS.

[0071] S7. Add 0.04% trypsin solution by volume at a 1:13 (g:mL) mass-to-volume ratio of the precipitate. Place the tube on a shaker at 160 rpm for 2 hours, then centrifuge at 8500 g for 9 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0072] S8. Place the decellularized precipitate in a vacuum freeze-drying device, evacuate to a pressure below 50 Pa, maintain the temperature at -58°C, dry for 16 hours, and then sterilize with ultraviolet light to obtain a sterile adipose tissue extracellular matrix.

[0073] S9. Grind 8 mg of sterile adipose tissue extracellular matrix into powder, add 1 mL of 0.012 mol / L HCl and 2.5 mg of trypsin and mix well. The final concentration of sterile adipose tissue extracellular matrix in the mixture is 8 mg / mL. Place it on a shaker at 250 rpm for 48 hours for digestion until no particles are visible to the naked eye. Obtain the digested sterile extracellular matrix sol and refrigerate it at 4°C.

[0074] S10. Add 10 mg of mesoporous molecular sieve SBA-15 to the digested sterile extracellular matrix sol, place it on a shaker at 400 rpm for 1 hour, and then centrifuge it at 4000 g for 10 minutes. Discard the precipitate and retain the extracellular matrix on the top layer.

[0075] S11. Adjust the pH value of the sterile extracellular matrix sol to 7.0 with 0.1 mol / L NaOH solution, and adjust the buffer salt concentration to 0.04 mol / L with 0.1 mol / L PBS to obtain a sterile injectable pregel, which was stored at 4°C.

[0076] Example 4 S1. Obtain allogeneic adipose tissue using resonance liposuction. Wash the adipose tissue at least three times with sterile saline until no blood or other tissue particles are visible. Centrifuge the washed adipose tissue at 3500 g for 7 minutes. Discard the upper lipid layer and lower liquid layer, and collect the middle adipose tissue layer.

[0077] S2. The collected adipose tissue layer was mechanically crushed using a tissue grinder. After weighing, sterile distilled water was added according to a mass-to-volume ratio of 1:10 (g:mL) after crushing. The mixture was centrifuged at a centrifugal force of 5000 g for 8 minutes. The upper layer of fat was discarded, and the lower layer of tissue was retained. PBS was added for ultrasonic cleaning at an ultrasonic frequency of 90 kHz for 1.8 hours to clean the obtained tissue layer.

[0078] S3. Add degreasing agent isopropanol according to the mass volume ratio of the crushed adipose tissue at 1:10 (g:mL). Place on a shaker at 150 rpm for 2 hours, then centrifuge at 8500 g for 9 minutes. Discard the degreasing agent, retain the precipitate, and wash with PBS.

[0079] S4. Add 0.1 mol / L NaOH solution at a mass-to-volume ratio of 1:10 (g:mL) to the precipitate. Place the precipitate on a shaker at 150 rpm for 2 hours, then centrifuge at 8500 g for 9 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0080] S5. Add 1% SDS solution by volume at a 1:10 (g:mL) mass-to-volume ratio of the precipitate. Place the tube on a shaker at 150 rpm for 2 hours, then centrifuge at 8500 g for 9 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0081] S6. Add 1% Triton X-100 solution by volume at a 1:10 (g:mL) mass-to-volume ratio of the precipitate. Place the tube on a shaker at 150 rpm for 2 hours, then centrifuge at 8500 g for 9 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0082] S7. Add 0.05% trypsin solution by volume at a 1:10 (g:mL) mass-to-volume ratio of the precipitate. Place the tube on a shaker at 150 rpm for 2 hours, then centrifuge at 8500 g for 9 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0083] S8. Place the decellularized precipitate in a vacuum freeze-drying device, evacuate to a pressure below 50 Pa, maintain the temperature at -50°C, dry for 15 hours, and then sterilize with ultraviolet light to obtain a sterile adipose tissue extracellular matrix.

[0084] S9. Grind 10 mg of sterile adipose tissue extracellular matrix into powder, add 1 mL of 0.01 mol / L HCl and 1 mg of pepsin and mix well. The final concentration of sterile adipose tissue extracellular matrix in the mixture is 10 mg / mL. Place the mixture on a shaker at 300 rpm for 48 hours for digestion until no particles are visible to the naked eye. Obtain the digested sterile extracellular matrix sol and refrigerate at 4°C.

[0085] S10. Add 10 mg of mesoporous molecular sieve SBA-15 to the digested sterile extracellular matrix sol, place it on a shaker at 350 rpm for 1.2 hours, and then centrifuge it at 4000 g for 10 minutes. Discard the precipitate and retain the extracellular matrix on the top layer.

[0086] S11. Adjust the pH value of the sterile extracellular matrix sol to 7.2 with 0.1 mol / L NaOH solution, and adjust the buffer salt concentration to 0.01 mol / L with 0.1 mol / L PBS to obtain a sterile injectable pregel, which was stored at 4°C.

[0087] Example 5 S1. Obtain human allogeneic adipose tissue using a combination of conventional vacuum liposuction and ultrasonic liposuction. Wash the adipose tissue at least three times with sterile saline until no blood or other tissue particles are visible. Centrifuge the washed adipose tissue at 3200 g for 6.5 minutes. Discard the upper lipid layer and lower liquid layer, and collect the middle adipose tissue layer.

[0088] S2. The collected adipose tissue layer was mechanically crushed using a tissue grinder. After weighing, sterile distilled water was added according to the mass-to-volume ratio of the crushed adipose tissue of 1:10 (g:mL). The adipose tissue was centrifuged at a centrifugal force of 4800 g for 7.5 minutes. The upper layer of fat was discarded, and the lower layer of tissue was retained. PBS was added for ultrasonic cleaning at an ultrasonic frequency of 30 kHz for 1 hour to clean the obtained tissue layer.

[0089] S3. Add degreasing agent isopropanol according to the mass volume ratio of the crushed adipose tissue at 1:10 (g:mL). Place on a shaker at 140 rpm for 2.2 hours, then centrifuge at 8200 g for 8.5 minutes. Discard the degreasing agent, retain the precipitate, and wash with PBS.

[0090] S4. Add 0.9 mol / L NaOH solution at a mass-to-volume ratio of 1:10 (g:mL) to the precipitate. Place the precipitate on a shaker at 140 rpm for 2.2 hours, then centrifuge at 8200 g for 8.5 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0091] S5. Add 0.8% SDS solution by volume at a 1:10 (g:mL) mass-to-volume ratio of the precipitate. Place the tube on a shaker at 140 rpm for 2.2 hours, then centrifuge at 8200 g for 8.5 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0092] S6. Add 1% Triton X-100 solution by volume at a 1:10 (g:mL) mass-to-volume ratio of the precipitate. Shake the tube at 150 rpm for 2.2 hours, then centrifuge at 8200 g for 8.5 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0093] S7. Add 0.05% trypsin solution by volume at a 1:10 (g:mL) mass-to-volume ratio of the precipitate. Place the tube on a shaker at 140 rpm for 2.2 hours, then centrifuge at 8200 g for 8.5 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0094] S8. Place the decellularized precipitate in a vacuum freeze-drying device, evacuate to a pressure below 50 Pa, maintain the temperature at -52°C, and dry for 16.5 hours. Then sterilize with ultraviolet light to obtain a sterile adipose tissue extracellular matrix.

[0095] S9. Grind 11 mg of sterile adipose tissue extracellular matrix into powder, add 1 mL of 0.011 mol / L HCl and 1.5 mg / mL pepsin and mix well. The final concentration of sterile adipose tissue extracellular matrix in the mixture is 11 mg / mL. Place the mixture on a shaker at 280 rpm for 50 hours for digestion until no particles are visible to the naked eye. Obtain the digested sterile extracellular matrix sol and refrigerate at 4°C.

[0096] S10. Add 12 mg of mesoporous molecular sieve SBA-15 to the digested sterile extracellular matrix sol, place it on a shaker at 350 rpm for 1.2 hours, and then centrifuge it at 3500 g for 8.5 minutes. Discard the precipitate and retain the extracellular matrix on the top.

[0097] S11. Adjust the pH value of the sterile extracellular matrix sol to 7.1 with 0.1 mol / L NaOH solution, and adjust the buffer salt concentration to 0.02 mol / L with 0.1 mol / L PBS to obtain a sterile injectable pregel, which was stored at 4°C.

[0098] Example 6 S1. Obtain human allogeneic adipose tissue using a combination of hydrodynamic liposuction and resonance liposuction. Wash the adipose tissue at least three times with sterile saline until no blood or other tissue particles are visible. Centrifuge the washed adipose tissue at 3800 g for 7 minutes. Discard the upper lipid layer and lower liquid layer, and collect the middle adipose tissue layer.

[0099] S2. The collected adipose tissue layer was mechanically crushed using a tissue grinder. After weighing, sterile distilled water was added according to a mass-to-volume ratio of 1:10 (g:mL) after crushing. The mixture was centrifuged at a centrifugal force of 5200 g for 8 minutes. The upper layer of fat was discarded, and the lower layer of tissue was retained. PBS was added for ultrasonic cleaning at a frequency of 75 kHz for 1.1 hours to clean the obtained tissue layer.

[0100] S3. Add degreasing agent isopropanol according to the mass volume ratio of the crushed adipose tissue at 1:10 (g:mL). Place on a shaker at 160 rpm for 2.5 hours, then centrifuge at 8800 g for 9 minutes. Discard the degreasing agent, retain the precipitate, and wash with PBS.

[0101] S4. Add 1.05 mol / L NaOH solution at a mass-to-volume ratio of 1:10 (g:mL) to the precipitate. Place the precipitate on a shaker at 160 rpm for 2.5 hours, then centrifuge at 8800 g for 9 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0102] S5. Add 0.9% SDS solution by volume at a 1:10 (g:mL) mass-to-volume ratio of the precipitate. Place the tube on a shaker at 160 rpm for 2.5 hours, then centrifuge at 8800 g for 9 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0103] S6. Add 0.85% Triton X-100 solution by volume at a 1:10 (g:mL) mass-to-volume ratio of the precipitate. Place the tube on a shaker at 160 rpm for 2.5 hours, then centrifuge at 8800 g for 9 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0104] S7. Add 0.06% trypsin solution by volume at a 1:10 (g:mL) mass-to-volume ratio of the precipitate. Place the tube on a shaker at 160 rpm for 2.5 hours, then centrifuge at 8800 g for 9 minutes. Remove the supernatant, retain the precipitate, and wash with PBS.

[0105] S8. Place the decellularized precipitate in a vacuum freeze-drying device, evacuate to a pressure below 50 Pa, maintain the temperature at -55°C, and dry for 17 hours. Then sterilize with ultraviolet light to obtain a sterile adipose tissue extracellular matrix.

[0106] S9. Grind 9 mg of sterile adipose tissue extracellular matrix into powder, add 1 mL of 0.013 mol / L HCl and 2 mg / mL pepsin and mix well. The final concentration of sterile adipose tissue extracellular matrix in the mixture is 9 mg / mL. Place the mixture on a shaker at 260 rpm for 45 hours for digestion until no particles are visible to the naked eye. Obtain the digested sterile extracellular matrix sol and refrigerate at 4°C.

[0107] S10. Add 16 mg of mesoporous molecular sieve SBA-15 to the digested sterile extracellular matrix sol, place it on a shaker at 250 rpm for 1 hour, and then centrifuge it at 4800 g for 9.5 minutes. Discard the precipitate and retain the extracellular matrix on the top layer.

[0108] S11. Adjust the pH value of the sterile extracellular matrix sol to 7.4 with 0.1 mol / L NaOH solution, and adjust the buffer salt concentration to 0.03 mol / L with 0.1 mol / L PBS to obtain a sterile injectable pregel, which was stored at 4°C.

[0109] Comparative Example 1 Traditionally, enzymes used in the preparation of extracellular matrix are inactivated by heating. While this procedure allows for precise control of the degree of degradation by measuring the duration of the enzyme reaction, the proteins remaining after enzyme denaturation and inactivation can still become allergens, posing unnecessary risks to the filler material.

[0110] Some preparation methods attempt to remove residual proteins after enzyme denaturation and inactivation by using dialysis to filter out impurities of a specific molecular weight to mitigate the risk of allergic reactions. However, dialysis has been found to be time-consuming, significantly increasing the time cost of extracellular matrix production.

[0111] The preparation method of the present invention uses mesoporous molecular sieve SBA-15 instead of dialysis treatment, and the advantages and disadvantages of the two treatment processes are verified by comparison.

[0112] The control product is also a self-crosslinked adipose decellularized matrix hydrogel, and its preparation method is as follows: Steps S1 to S9 and S11 are consistent with the method provided in Example 6. The difference is S10: S10. Transfer the digested sol to a 50 kDa sterile MWCO dialysis bag, tie the bag tightly, and place it in a 4°C pre-cooled sterile PBS solution with a dialysate volume 100 times the sol volume for 24 hours. Change the dialysate every 6 hours. After dialysis, remove the sol from the dialysis bag and centrifuge it at a centrifugal force of 4800 g for 9.5 minutes. Discard the precipitate and retain the upper sterile extracellular matrix sol.

[0113] The hydrogel prepared in Comparative Example 1 and the hydrogel obtained in Example 6 were subjected to ELISA test, and the residual pepsin content therein was measured as shown in Table 1.

[0114] Table 1. ELISA test results

[0115] By comparing the test results, it can be seen that the effect of using mesoporous molecular sieves to remove pepsin is close to that of using dialysis bags, and the residual amount is less than 10%, which meets the product safety requirements. However, from the perspective of the preparation method, the use of mesoporous molecular sieves only requires shaking on a shaking table for 1 hour, while the use of dialysis bags requires a dialysis time of up to 24 hours, and the dialysate needs to be replaced 3 times. In addition, the mesoporous molecular sieve after use can be heated and ultrasonically treated with SDS solution to re-elute the enzyme, thereby achieving reuse and further reducing production costs. However, since the dialysis bag is in contact with perishable samples such as pepsin or trypsin, the residual substances may clog the membrane pores or cause degradation, affecting the dialysis efficiency and even causing cross contamination, so it can only be used once. It can be seen that the preparation method of the fat decellularized matrix self-crosslinking hydrogel provided by the present invention has the advantages of low cost and high economic benefit under the premise of ensuring the low allergenicity of the hydrogel.

[0116] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

Claims

1. A method for preparing a self-crosslinked adipose decellularized matrix hydrogel, characterized in that: Including steps: S1. Collect human adipose tissue, wash it at least three times with cleaning solution, centrifuge and discard the upper fat layer and lower liquid layer, and collect only the middle adipose tissue layer; S2, mechanically disrupting the adipose tissue layer, adding sterile distilled water for centrifugation, discarding the upper layer of fat, retaining the lower layer of tissue, and then adding a cleaning solution for ultrasonic cleaning; S3. Add degreasing reagent to the cleaned tissue layer and shake it, then centrifuge it and retain the precipitate; S4, replacing the delipidation reagent with a decellularization reagent, and repeating step S3; S5. The decellularized precipitate is freeze-dried and sterilized to obtain a sterile adipose tissue extracellular matrix; S6, grinding the sterile adipose tissue extracellular matrix, adding a digestion reagent, mixing, and shaking to digest until no particles are visible to the naked eye, to obtain a sol; S7, adding mesoporous molecular sieves to the sol, shaking to absorb excess digestion reagent, and centrifuging to remove precipitates to obtain extracellular matrix; S8. Use a neutralizing reagent pre-frozen at 4°C to adjust the pH value of the extracellular matrix to 6.8-7.5, and add a buffer reagent pre-frozen at 4°C to adjust the buffer salt concentration to obtain a sterile injectable pregel, which is stored at 4°C.

2. The preparation method according to claim 1, characterized in that The working parameters of the centrifugation in step S1 are 2000~4000 g, and the centrifugation time is 5~8 minutes; preferably, the working parameters of the centrifugation in step S2 are 3000~6000 g, and the centrifugation time is 6~10 minutes; preferably, the working parameters of the centrifugation in steps S3 and S4 are 7000~10000 g, and the centrifugation time is 8~10 minutes; preferably, the working parameters of the centrifugation in step S7 are 3000~5000 g, and the centrifugation time is 8~10 minutes.

3. The preparation method according to claim 1, characterized in that: The decellularization reagent includes a decellularization reagent A, a decellularization reagent B, a decellularization reagent C and a decellularization reagent D, wherein the decellularization reagent A is selected from one of sodium chloride, hydrochloric acid or sodium hydroxide, the decellularization reagent B is selected from sodium lauryl sulfate or sodium deoxycholate, the decellularization reagent C is selected from one of Triton X-100 or 3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt, and the decellularization reagent D is selected from trypsin or pepsin.

4. The preparation method according to claim 1 or 3, characterized in that The ratio of the tissue layer to the degreasing reagent is 1 g: (8-15) mL; the ratio of the precipitate to the decellularization reagent is 1 g: (8-15) mL.

5. The preparation method according to claim 1, characterized in that: The ultrasonic cleaning process has a working parameter of 20-100 kHz and a cleaning time of 1-2 hours.

6. The preparation method according to claim 1, characterized in that: The degreasing agent is selected from one of methanol, isopropanol or anhydrous ethanol.

7. The preparation method according to claim 1, characterized in that: The freeze-drying process parameters are vacuuming to below 50 Pa, temperature -40 to -60°C, and time of 10 to 20 hours.

8. The preparation method according to claim 1, characterized in that: The digestion reagent includes digestion reagent A and digestion reagent B, wherein the digestion reagent A is selected from hydrochloric acid or acetic acid, and the digestion reagent B is selected from trypsin or pepsin.

9. A self-crosslinked adipose decellularized matrix hydrogel, characterized in that: Prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the adipose decellularized matrix self-crosslinking hydrogel according to claim 9 in medical filling materials.

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