A hydrophilic acellular matrix scaffold from a fat source, its preparation method and application
Through the enzyme-free method and rinsing method of treraton X-100 solution, the problem of cumbersome preparation process and high cost in the prior art is solved, and more efficient fat-forming effect and cost reduction are achieved.
Patent Information
- Application Number
- CN202411166625.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-23
- Publication Date
- 2025-06-13
- Estimated Expiration
- 2044-08-23
AI Technical Summary
The prior art lacks targeted optimization when preparing fat-derived decellular matrix scaffolds, which are costly, cumbersome and time-consuming, resulting in loss of beneficial components and damage to physical structure.
Using enzyme-free method, chemical rinsing was performed through traluton X-100 solution to retain more protein components and washed in the last step with ultrapure water to ensure that the hydrophilic beneficial protein is not lost, and the isopropanol degreasing step is omitted.
It improves the fat-forming effect of the scaffold, reduces the preparation cost, simplifies the process, shortens the rinsing time, and retains more beneficial ingredients such as growth factors.
Smart Images
Figure CN119075007B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomaterials, and particularly relates to a hydrophilic adipose-derived acellular matrix scaffold, a preparation method thereof, and an application thereof. Background Art
[0002] Adipose-derived acellular matrix (DAM) biologic scaffolds can achieve adipose tissue regeneration in vivo and are used for soft tissue reconstruction, with broad clinical application prospects. Since Flynn proposed the classic DAM preparation method in 2010, more than a dozen improved and optimized methods have been reported, but the following problems mainly exist in each method:
[0003] 1. The optimization of preparation lacks pertinence. All preparation processes include steps such as cell disruption, removal of cell components, and removal of lipid components. There is no targeted optimization method designed for retaining a certain beneficial component.
[0004] 2. The preparation cost is high. Classic decellularization methods require various biological enzymes (trypsin, ribonuclease, deoxyribonuclease, lipase), etc. Although the total decellularization time is greatly shortened, the cost of enzymes is very high, and the enzymes are derived from cattle, sheep, etc., increasing the risk of introducing xenogeneic antigens.
[0005] 3. The preparation process is cumbersome and time-consuming. The classic decellularization method takes 109 hours, and various washing reagents and enzymes are repeatedly used, with cumbersome steps. Long-time rinsing will lead to the loss of beneficial components and damage the physical structure of the scaffold. Among various optimization methods, the enzyme-free method omits the use of enzymes and shortens the preparation time to a certain extent, but all also require more than three days. Summary of the Invention
[0006] To solve the deficiencies of the prior art, the present invention provides a hydrophilic adipose-derived acellular matrix scaffold, a preparation method thereof, and an application thereof. On the basis of retaining the beneficial components in DAM, that is, hydrophilic components, the present invention proposes a new technical solution for decellularization based on the enzyme-free method.
[0007] The technical solution provided by the present invention is as follows:
[0008] A preparation method of a hydrophilic adipose-derived acellular matrix scaffold, comprising the following steps:
[0009] 1) Obtain a hydrophilic adipose-derived acellular matrix precursor from physically disrupted adipose tissue;
[0010] 2) Chemically rinse the hydrophilic adipose-derived acellular matrix precursor with Triton X-100 solution to obtain a supernatant and a precipitate;
[0011] 3) Separate the precipitate obtained in step 2), wash it with ultrapure water, and collect the precipitate to obtain a hydrophilic adipose-derived acellular matrix scaffold.
[0012] In the above technical solution:
[0013] Through proteomic analysis, it can be known that chemical rinsing with Triton can retain more protein components compared with rinsing with NaCl aqueous solution in the first step by the enzyme-free method;
[0014] Secondly, set the long-time water rinsing in the last step. At this time, the hydrophilic proteins of DAM have wound into flocs and are insoluble in water. Rinsing with water will not lose the beneficial hydrophilic proteins;
[0015] Finally, since it is a hydrophilic protein and does not contain lipid components, the step of defatting with isopropanol can be omitted in this technical solution.
[0016] This is the fundamental reason why the scaffold prepared by this process has excellent adipogenic effects.
[0017] Specifically, in step 1): The adipose tissue is discarded liposuction adipose tissue.
[0018] Based on the above technical solution, the discarded liposuction adipose tissue can be utilized.
[0019] Specifically, in step 1), the hydrophilic adipose-derived acellular matrix precursor is: the hydrophilic component obtained by separating oil and lipophilic components from the fat slurry formed by physical fragmentation.
[0020] The hydrophilic component can be separated by existing means based on the separation principle of hydrophilic and lipophilic components, or the following method can be used:
[0021] 1. Preparation of the scaffold donor:
[0022] Collect 200 ml of discarded liposuction adipose tissue from the clinic as the donor. After picking out the thick fibrous tissue, fascia, etc., divide it into 50 ml centrifuge tubes, with 25 ml of adipose tissue in each tube. Add 25 ml of PBS, soak it, invert it 5 times to shake well, and centrifuge at low speed (500 G, 2 min) to remove grease, blood, swelling fluid, etc., and retain the middle fat layer. Repeat the PBS washing three times.
[0023] 2. Physical fragmentation:
[0024] Collect the washed adipose tissue and centrifuge it again to remove as much water as possible. Homogenize it with a homogenizer (FJ200-SH, Huxi Ltd., Shanghai, China) at high speed (20,000 r / min) for 2 minutes until it becomes completely liquid without visible particulate components to the naked eye.
[0025] 3. Separation of hydrophilic and lipophilic components: Centrifuge each 50 ml of physically disrupted adipose liquid at high speed (3500 G, 3 min). The precipitate is the hydrophilic DAM precursor component, and the volume of the hydrophilic DAM precursor obtained is 3.5 ml.
[0026] Specifically, step 2) includes the following steps:
[0027] 2a) Add hydrophilic adipose-derived decellularized matrix precursor and triton X-100 solution to a centrifuge tube;
[0028] 2b) Shake on a shaker;
[0029] 2c) Centrifuge at high speed, remove the supernatant, and add a new triton X-100 solution to the precipitate part; or filter the liquid component through a filter mesh and add a new triton X-100 solution to the collected solid component;
[0030] 2d) Repeat steps 2b) to 2c) at least twice to obtain a supernatant and a precipitate.
[0031] Specifically, in step 2a), the volume concentration of the triton X-100 solution is 0.8 - 1.2%; the volume ratio of the hydrophilic adipose-derived decellularized matrix precursor to the triton X-100 solution is 1:(9 - 11).
[0032] Specifically, in step 2b), the operating temperature is 36 - 38 °C; the operating time is 4.5 - 5.5 hours.
[0033] Specifically, in step 2c), the centrifugal force for high-speed centrifugation is 3400 - 3600 G, the time is 2.5 - 3.5 min; the volume concentration of the new triton X-100 solution is 0.8 - 1.2%.
[0034] Specifically, in step 2c), repeat steps 2b) to 2c) twice to obtain a supernatant and a precipitate.
[0035] Specifically, step 3) includes the following steps:
[0036] 3a) Centrifuge at high speed to remove the supernatant, add ultrapure water to the precipitate, and shake on a shaker; or filter the liquid component through a filter mesh and add new ultrapure water to the collected solid component;
[0037] 3b) Centrifuge at high speed to remove the supernatant, add ultrapure water to the precipitate for the second time, and shake on a shaker.
[0038] 3c) Centrifuge at high speed to remove the supernatant, add ultrapure water to the precipitate for the third time, and shake on a shaker.
[0039] 3d) Centrifuge and collect the precipitate to obtain a hydrophilic acellular matrix scaffold from a fat source.
[0040] Specifically, in step 3a), the volume ratio of the hydrophilic fat-derived acellular matrix precursor to ultrapure water is 1:(9 - 11); the temperature of shaking on the shaker is 36 - 38 °C, and the time is 0.4 - 0.6 hours.
[0041] Specifically, in step 3b), the volume ratio of the hydrophilic fat-derived acellular matrix precursor to ultrapure water is 1:(9 - 11); the temperature of shaking on the shaker is 36 - 38 °C, and the time is 6 - 9 hours.
[0042] Specifically, in step 3c), the volume ratio of the hydrophilic fat-derived acellular matrix precursor to ultrapure water is 1:(9 - 11); the temperature of shaking on the shaker is 36 - 38 °C, and the time is 0.4 - 0.6 hours.
[0043] The present invention also provides a hydrophilic acellular matrix scaffold prepared according to the above scheme.
[0044] Specifically, the total content of four key cytokines, bFGF, VEGF, CXCL12, and CCL21, in the hydrophilic acellular matrix scaffold from a fat source is greater than 1 ng / mL.
[0045] The present invention also provides the application of the hydrophilic acellular matrix scaffold from a fat source for preparing a fat regeneration biological scaffold.
[0046] The beneficial effects of the present invention are as follows:
[0047] 1. Provide the preparation steps suitable for hydrophilic DAM, effectively improving the adipogenic effect of the scaffold (refer to Figure 3 ).
[0048] 2. Reduce the preparation cost. Only a 1% dilution of Triton X-100 solution is used throughout the process, and the cost is greatly reduced compared with the classical method using enzymes.
[0049] 3. Simplify the process. The entire rinsing process of the preparation only takes 25 hours. The short-time rinsing ensures the effective removal of DNA (refer to Figure 5 ), and at the same time retains more beneficial components, such as growth factors (refer to Figure 6 ). Brief Description of the Drawings
[0050] Figure 1 It is the effect diagram after adding 1% triton X-100 solution in Example 1.
[0051] Figure 2 It is the hydrophilic DAM scaffold diagram obtained in Example 1.
[0052] Figure 3 It is the comparison diagram of the adipogenic induction effect after 3 weeks of implanting the hydrophilic scaffold prepared by the prior art and the hydrophilic scaffold provided in Example 1 of the present invention into nude mice.
[0053] Figure 4 It is the comparison diagram of the protein abundance eluted by NaCl aqueous solution and 1% triton X-100 solution.
[0054] Figure 5 It is the comparison diagram of the DNA residue amount in the hydrophilic scaffold prepared by the prior art and the hydrophilic scaffold provided in Example 1 of the present invention.
[0055] Figure 6 It is the comparison diagram of the cytokine content in the hydrophilic scaffold prepared by the prior art and the hydrophilic scaffold provided in Example 1 of the present invention. Detailed implementation mode
[0056] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.
[0057] Unless otherwise specified, the test methods used in the invention examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.
[0058] Example 1
[0059] 1. Isolate 7 ml of hydrophilic DAM precursor components from the physically disrupted adipose tissue.
[0060] 2. Divide into two tubes for chemical rinsing:
[0061] 2.1. Add 3.5 ml of hydrophilic DAM and 30 ml of 1% triton X-100 solution (volume ratio 1:10, do not fill up to leave sufficient space for shaking) into a 50 ml centrifuge tube (as Figure 1 shown).
[0062] 2.2. Shake on a shaker at 37 °C for 5 hours.
[0063] 2.3. Centrifuge at high speed (3500 G, 3 min), remove the supernatant, and add 30 ml of new 1% triton X-100 to the precipitate part.
[0064] 2.4. Repeat steps 2.2 - 2.3 three times in total, for 15 hours in total.
[0065] 3. Ultra-pure water washing:
[0066] 3.1. After high-speed centrifugation, remove the supernatant (1% tritonX-100), add 30 ml of ultra-pure water to the precipitate, and shake on a shaker at 37°C for 0.5 hour.
[0067] 3.2. After high-speed centrifugation, remove the supernatant, add 30 ml of ultra-pure water to the precipitate, and shake on a shaker at 37°C for 7.5 hours.
[0068] 3.3. After high-speed centrifugation, remove the supernatant, add 30 ml of ultra-pure water to the precipitate, and shake on a shaker at 37°C for 0.5 hour.
[0069] 3.4. Centrifuge and collect the precipitate as the hydrophilic DAM scaffold, 6.5 mL in two tubes (as Figure 2 shown).
[0070] Comparative Example 1
[0071] Refer to the preparation method of Example 1, the difference is that the concentration of the tritonX-100 solution is adjusted down to 0.5%, and the result is that steps 2.2 - 2.3 need to be repeated at least five times before the rinsing solution becomes clear. The operation steps and time are increased.
[0072] Comparative Example 2
[0073] Refer to the preparation method of Example 1, the difference is that the concentration of the tritonX-100 solution is adjusted up to 2%, steps 2.2 - 2.3 need to be repeated three times, and the result is that the time of the subsequent ultra-pure water washing step is significantly increased.
[0074] Effect Example
[0075] For comparison, the "enzyme-free method" of the following existing technology is used to prepare the hydrophilic scaffold:
[0076] 1. Immerse the hydrophilic DAM precursor in a 0.5 M NaCl solution and shake on a shaker at 37°C for 4 hours. After centrifuging at 3500G for 3 min, pour off the supernatant.
[0077] 2. Immerse the precipitate in a 1 M NaCl solution and shake on a shaker at 37°C for 4 hours. After centrifuging at 3500G for 3 min, pour off the supernatant.
[0078] 3. Immerse the precipitate in ultra-pure water and shake on a shaker at 37°C overnight. After centrifuging at 3500G for 3 min, pour off the supernatant.
[0079] 4. The precipitate was immersed in 1% tritonX-100 solution and shaken on a shaker at 37°C for 72 hours. The solution was changed every 8 hours. Each time the solution was changed, it was centrifuged at 3500G for 3 minutes, and the supernatant was discarded.
[0080] 5. The precipitate was immersed in ultrapure water and shaken on a shaker at 37°C for half an hour. After centrifuging at 3500G for 3 minutes, the supernatant was discarded. This was repeated twice.
[0081] 6. The precipitate was immersed in 99% isopropanol and shaken on a shaker at 37°C for 8 hours. After centrifuging at 3500G for 3 minutes, the supernatant was discarded.
[0082] 7. Repeat step 5. The obtained precipitate is a hydrophilic scaffold prepared by the prior art.
[0083] As Figure 3 shown, it is a comparison diagram of the adipogenic induction effect after implanting the hydrophilic scaffold prepared by the prior art and the hydrophilic scaffold provided in Example 1 of the present invention into nude mice for 3 weeks. It can be seen that the area of adipocytes induced by the hydrophilic scaffold provided in Example 1 of the present invention is significantly larger than that of the existing hydrophilic scaffold. The scale bar in the figure is 400um.
[0084] As Figure 4 shown, it is a comparison diagram of the protein abundance eluted by NaCl aqueous solution and 1% tritonX-100 solution. It can be seen that more protein components were eluted by the NaCl aqueous solution than by the tritonX-100 solution for the chemical rinsing of the hydrophilic DAM precursor components.
[0085] As Figure 5 shown, it is a comparison diagram of the residual DNA content data in the hydrophilic scaffold prepared by the prior art and the hydrophilic scaffold provided in Example 1 of the present invention. It can be seen that there is no significant difference in the residual DNA content between the existing hydrophilic scaffold prepared by the enzyme-free method and the hydrophilic scaffold prepared by Example 1 of the present invention, and both meet the decellularization standard.
[0086] As Figure 6 shown, it is a comparison diagram of the cytokine content in the hydrophilic scaffold prepared by the prior art and the hydrophilic scaffold provided in Example 1 of the present invention. It can be seen that the content of beneficial cytokines that can be retained by the hydrophilic scaffold provided by the present invention is significantly greater than that of the existing scaffold.
[0087] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.
Claims
1. A method for preparing a hydrophilic fat-derived acellular matrix scaffold, characterized in that: The following steps are involved: 1) Obtaining hydrophilic adipose-derived acellular matrix precursor from physically disrupted adipose tissue; 2) chemically rinsing the hydrophilic adipose-derived acellular matrix precursor with tritonX-100 solution to obtain a supernatant and a precipitate; 3) separating the precipitate obtained in step 2), washing it with ultrapure water, collecting the precipitate, and obtaining a hydrophilic fat-derived acellular matrix scaffold; In step 1), the hydrophilic fat-derived acellular matrix precursor is: a hydrophilic component obtained by separating oil and lipophilic components from a fat slurry formed by physical crushing; Step 2) includes the following steps: 2a) Add hydrophilic adipose-derived acellular matrix precursor and tritonX-100 solution into a centrifuge tube; 2b) Shake on a shaker at a temperature of 36-38°C and a time of 4.5-5.5 hours; 2c) High-speed centrifugation, remove the supernatant, add new tritonX-100 solution to the precipitate, the centrifugal force of high-speed centrifugation is 3400-3600G, and the time is 2.5-3.5min; 2d) Repeat steps 2b) to 2c) at least twice to obtain a supernatant and a precipitate; Step 3) includes the following steps: 3a) Remove the supernatant by high-speed centrifugation, add ultrapure water to the precipitate, and shake on a shaker; 3b) Remove the supernatant by high-speed centrifugation, add ultrapure water to the precipitate for the second time, and shake on a shaker; 3c) Remove the supernatant by high-speed centrifugation, add ultrapure water to the precipitate for the third time, and shake on a shaker; 3d) Centrifuge and collect the precipitate to obtain the hydrophilic fat-derived acellular matrix scaffold.
2. The method for preparing the hydrophilic fat-derived acellular matrix scaffold according to claim 1, characterized in that: In step 1), the adipose tissue is discarded liposuction adipose tissue.
3. The method for preparing the hydrophilic fat-derived acellular matrix scaffold according to claim 1, characterized in that: In step 2a), the volume concentration of tritonX-100 solution is 0.8-1.2%; the volume ratio of hydrophilic adipose-derived acellular matrix precursor to tritonX-100 solution is 1:(9-11); In step 2c), the volume concentration of the new tritonX-100 solution is 0.8 to 1.2%; In step 2c), steps 2b) to 2c) are repeated twice to obtain a supernatant and a precipitate.
4. The method for preparing the hydrophilic fat-derived acellular matrix scaffold according to claim 1, characterized in that: In step 3a), the volume ratio of the hydrophilic adipose-derived acellular matrix precursor to ultrapure water is 1:(9-11); the shaking temperature is 36-38° C., and the shaking time is 0.4-0.6 hours; In step 3b), the volume ratio of the hydrophilic adipose-derived acellular matrix precursor to ultrapure water is 1:(9-11); the shaking temperature is 36-38° C., and the shaking time is 6-9 hours; In step 3c), the volume ratio of the hydrophilic adipose-derived acellular matrix precursor to ultrapure water is 1:(9-11); the shaking temperature is 36-38° C., and the shaking time is 0.4-0.6 hours.
5. A hydrophilic fat-derived acellular matrix scaffold prepared according to the preparation method according to any one of claims 1 to 4.
6. The hydrophilic adipose-derived acellular matrix scaffold according to claim 5, characterized in that: The total content of four cytokines, bFGF, VEGF, CXCL12 and CCL21, was greater than 1 ng / mL.
7. An application of the hydrophilic fat-derived acellular matrix scaffold according to claim 5 or 6, characterized in that: Used to prepare fat regeneration biological scaffolds.