Regenerated decellularized adipose derivative as well as preparation method and application thereof

Abstract

The invention relates to a regenerated decellularized adipose derivative as well as a preparation method and application thereof. The preparation raw material of the regenerated decellularized adipose derivative is prepared from a vascular matrix component (SVF) separated from human adipose tissue through decellularization treatment and freeze drying; the prepared regenerated decellularized adipose derivative is a porous material, the porosity is greater than 90%, and the aperture is 50-200 [mu] m; the composition comprises the following components in percentage by weight: 60-70% of collagen, 15-20% of elastin, 1 * 10 < 10 >-5 * 10 < 10 > particles / g of exosome, less than or equal to 5 ng / mg of DNA (deoxyribonucleic acid) residue, more than or equal to 30 ng / g of VEGF (vascular endothelial growth factor) and more than or equal to 15 ng / g of FGF-2 (fibroblast growth factor). The prepared regenerated decellularized adipose derivative is low in DNA residual quantity, rich in growth factors and capable of meeting the requirement for deep tissue filling due to the fact that the collagen content and the elastin content are high.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, and in particular to a regenerated decellularized fat derivative, its preparation method, and its application. Background Technology

[0002] Fat grafting, as a method of tissue repair and shaping using natural biomaterials, has been widely used in areas such as improving facial depressions, anti-aging, and contouring, and has shown promising application prospects.

[0003] However, existing fat grafting techniques still have many significant limitations in practical applications, mainly in the following aspects: (1) Regarding the source of donors and survival rate: Currently, only autologous fat can be used as a filling material. Due to the difference in the absorption rate of fat between the donor area and the recipient area, the survival rate of fat is unstable, and multiple transplants are usually required to achieve the desired effect. (2) Risks in the collection process: The collection of donor fat needs to be completed through minimally invasive liposuction, which involves anesthesia risks, and patients have a certain recovery period after the operation; (3) Processing time: The collected fat needs to undergo standardized processing procedures such as purification and centrifugation, which usually takes 2 to 4 hours, significantly prolonging the overall operation time; (4) Long-term effects and safety: The long-term retention rate of free fat particles is greatly affected by the operation technique, and there is a risk of complications such as calcified nodules or lumps. (5) Treatment cost: Compared with traditional fillers, fat grafting has a higher treatment cost.

[0004] To address the aforementioned problems, those skilled in the art have made various attempts and proposed different technical solutions, but these solutions still have their own shortcomings: For example, there are methods to prepare adipose-derived mesenchymal stem cell extracts for use as fat grafting materials, but the material solutions provided do not achieve complete decellularization and have a high residual DNA content (e.g., >50 ng / mg), which poses a risk of triggering an immune rejection reaction; Another approach uses traditional decellularized materials, but these materials have insufficient mechanical strength (e.g., modulus < 10 kPa) due to the easy degradation of collagen, and therefore cannot meet the needs of deep tissue filling. Another approach involves using a freeze-thaw method to decellularize adipose tissue. However, the final product treated with this method loses more than 80% of its growth factor activity compared to the raw material, which significantly limits the product's repair effect.

[0005] Therefore, there is an urgent need for a fat grafting technology or material that can overcome the above-mentioned defects while taking into account safety, mechanical properties, bioactivity, and ease of operation, in order to meet the needs of clinical applications. Summary of the Invention

[0006] The purpose of this invention is to disclose a regenerated decellularized lipid derivative, its preparation method and application, in order to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0007] The first aspect of the present invention is to provide a regenerated decellularized fat derivative.

[0008] A second aspect of the present invention is to provide a method for preparing the regenerated decellularized lipid derivative described in the first aspect of the present invention.

[0009] A third aspect of the present invention is to provide applications of the regenerated decellularized lipid derivatives described in the first aspect of the present invention.

[0010] The regenerated decellularized adipose-derived fat derivative of the first aspect of the present invention is prepared from vascular matrix component (SVF) isolated from human adipose tissue, which is then decellularized and freeze-dried. The obtained regenerated decellularized adipose-derived fat derivative is a porous material with a porosity >90% and a pore size of 50-200 μm. Its components, by weight percentage, include: collagen 60-70%, elastin 15-20%, and exosomes 1×10⁻⁶. 10 ~5×10 10 The regenerated acellular fat derivative has low DNA residue, is rich in growth factors, and has high collagen and elastin content, thus meeting the needs of deep tissue filling. The product contains particles / g, with DNA residue ≤5 ng / mg, vascular endothelial growth factor (VEGF) content ≥30 ng / g, and fibroblast growth factor 2 (FGF-2) content ≥15 ng / g.

[0011] The preparation method according to the second aspect of the present invention includes the following steps: S1) SVF was isolated from human adipose tissue and amplified to the 3rd to 5th generation under hypoxic conditions to obtain culture products; S2) The culture product was treated with 0.05-0.20% Triton X-100 and a weak alkaline solution to remove cells and cell debris, and a decellularized product was obtained. S3) Freeze-dry the decellularized product to obtain the regenerated decellularized lipid derivative.

[0012] Human adipose tissue is typically obtained from subcutaneous fat in the abdomen or thigh. The resulting SVF contains adipose-derived stem cells, endothelial cells, pericytes, and a small number of blood cells. The preparation method involves expanding and culturing the SVF in a hypoxic environment, causing the cells to produce more exomammary matrix, exosomes, and growth factors. In the decellularization step, treatment with a low concentration of Triton X-100 and a weak alkaline solution gently removes cells, ensuring low DNA residue while preventing the denaturation and inactivation of growth factors.

[0013] In a further application implementation, the amplification under hypoxic conditions refers to culture in DMEM / F12 medium containing 10% fetal bovine serum under conditions of 3% O2 ​​and 5% CO2.

[0014] In a further application embodiment, the method for isolating the SVF from human adipose tissue includes the following steps: 1) Obtain adipose tissue from a healthy human body, rinse 3-5 times with sterile saline to remove residual blood and impurities, and then mince the adipose tissue to 1-3 mm. 3 Fragments; 2) Add 0.075% type I collagenase solution (containing 1% penicillin-streptomycin), and digest at 37°C with constant temperature shaking at 120 rpm for 30-60 minutes; 3) Filter the tissue suspension using a 200-mesh filter, centrifuge the filtrate at 3000 g for 10-20 minutes at 4°C to remove the oil and digestion residues from the supernatant, and the precipitate is the SVF.

[0015] In a further application embodiment, the weak alkaline solution is 0.25% ammonia water.

[0016] In a further application embodiment, step S2) of processing the culture product includes the following steps: a) Add Triton X-100 to the culture product and shake for 1-2 hours; b) Add an equal volume of 0.25% ammonia solution and shake for 1-2 hours.

[0017] Stepwise processing can remove different components, resulting in very low DNA residue levels; the Triton X-100 concentration is far lower than the 1% used in conventional decellularization, reducing damage to the extracellular matrix (ECM); at the same time, the appropriate concentration of ammonia can avoid excessive alkaline hydrolysis of collagen.

[0018] In a further application implementation, the removal of cells and cell debris in step S2) is achieved by centrifugation and washing, specifically by centrifuging at 100~1000 g for 10~20 minutes and washing with PBS solution 3~5 times.

[0019] In a further application implementation, step S3) of freeze-drying includes the following steps: Pretreatment: Add a protective agent to the decellularized product; Pre-freeze: -80℃, maintain for 2~4 hours; Primary drying: Evacuate to 10 Pa, raise the temperature to -20~-10℃, and maintain until the ice crystals completely sublimate; Desorption and drying: Heat to 25~30℃ and maintain for 6~12 hours.

[0020] Primary drying mainly removes free water, while desorption drying removes bound water. Furthermore, the temperature during the desorption drying process should not be too high, otherwise the ECM may be denatured.

[0021] In a further application embodiment, the protective agent is selected from 5-10% (w / v) trehalose or 0.5-2% (v / v) glycerol. Adding a protective agent in the pretreatment step can reduce ice crystal formation and maintain the porosity of the matrix through vitrification.

[0022] In a further implementation, the pre-freezing step involves cooling from room temperature to -40°C at a rate of 1°C / minute, and then cooling to -80°C at a rate of 5°C / minute. The pre-freezing process cannot be too fast, otherwise large ice crystals will easily form, damaging the ECM; nor can it be too slow, consuming too much energy. The third aspect of the application of this invention refers to the use of the regenerated decellularized fat derivative in the preparation of soft tissue filler materials, wound repair materials or scar-inhibiting drugs, such as for filling facial depressions from the deep dermis to the subcutaneous fat layer or repairing diabetic foot ulcers.

[0023] The present invention has at least the following beneficial effects: 1. High Mechanical Strength: This invention achieves a significant improvement in the mechanical strength of the regenerated decellularized fat derivative by synergistically combining high ECM component retention, precise decellularization process, and porous freeze-drying structure design, while eliminating immunogenicity. Its compressive modulus far exceeds that of traditional soft tissue fillers, and the elasticity imparted by the porous structure gives it both support and flexibility in deep tissue filling.

[0024] 2. Long-lasting repair: The exosomes contained in the regenerated decellularized fat derivative have a sustained release period of more than 28 days (in vitro dialysis experimental data), which can promote angiogenesis and collagen remodeling. Attached Figure Description

[0025] Figure 1 This is a SEM image of the regenerated decellularized lipid derivative. Detailed Implementation

[0026] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention are within the scope of the present invention.

[0027] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0028] Example 1: Preparation of regenerated decellularized lipid derivatives 200 g of abdominal adipose tissue was extracted from a healthy donor (30 years old, BMI 22). The tissue was rinsed 3-5 times with sterile saline to remove residual blood and impurities. The adipose tissue was then minced to 1-3 mm. 3 Cell fragments were collected; 0.075% type I collagenase (Sigma-Aldrich) solution (containing 1% penicillin-streptomycin) was added, and the cells were digested at 37°C with constant shaking at 120 rpm for 60 minutes; the tissue suspension was filtered through a 200-mesh filter, and the filtrate was centrifuged at 4°C for 20 minutes to remove oil and digestion residues from the supernatant, yielding SVF. The obtained SVF was cultured in DMEM / F12 medium containing 10% fetal bovine serum under 3% O2 ​​and 5% CO2 conditions to passage 4 (7 days); 5 mL of 0.1% Triton X-100 solution was added to the SVF cell suspension and the cells were shaken for 2 hours, followed by 5 mL of 0.25% ammonia solution (pH 10.5) and shaken for 2 hours. The suspension was then centrifuged at 3000 g for 10 minutes to remove cell debris, and washed three times with PBS solution. Add 5% (w / v) trehalose to the cleaned decellularized matrix, place the sample in a freeze dryer, and lower the temperature from room temperature to -40°C at 1°C / min, then lower it to -80°C at 5°C / min, maintain for 2 hours, evacuate the freeze dryer to 10 Pa, raise the temperature to -10°C, maintain for 12 hours until the ice crystals completely sublimate, raise the temperature to 25~30°C, maintain for 6~12 hours, and the final product is the freeze-dried fiber of the desired regenerated decellularized lipid derivative.

[0029] Comparative Example 1: A freeze-dried powder of human mesenchymal stem cell extract was prepared using existing technology. The subcutaneous adipose tissue extract obtained by liposuction (after excluding endocrine diseases and infectious diseases) was centrifuged at 800 g, washed with PBS buffer, soaked, and washed three times at 800 g for 4 minutes each time. Visible blood vessels and connective tissue were removed, and the tissue was thoroughly minced with ophthalmic scissors and placed in a 50 mL centrifuge tube. Two volumes of 0.2% collagenase, one volume of 1% BSA, and antibiotics were added, mixed well, sealed, and digested at 37°C for 45 minutes until a paste-like consistency was reached. An equal volume of low-glucose DMEM containing 10% fetal bovine serum was added to terminate digestion. The tube was centrifuged at 1800 r / min for 10 minutes. After centrifugation, the tissue separated into three layers: the upper layer consisted of fat and incompletely digested adipose tissue; the middle layer was the supernatant; and the lower layer was a mixture of adipose-derived stem cells and erythrocytes. The upper and middle layers were discarded, and the cell pellet in the lower layer was resuspended in complete culture medium. The mixture was filtered through a 70 μm cell sieve, and the cell concentration of the filtrate was adjusted to 5 × 10⁶ cells / mL. 5 Mononuclear cells / mL were seeded at T-25cm 2 In a culture flask, culture at 37°C, 5% CO2, and saturated humidity. After 48 hours, change the medium and add complete adipose-derived stem cell culture medium to continue culturing.

[0030] Identified passaged human adipose-derived mesenchymal stem cells (P3-P30 generations) were cultured in cell growth medium (prepared by adding 10% fetal bovine serum, 200 U / mL penicillin, and 250 U / mL streptomycin to commercially available basic DMEM / F12 culture medium) until 80% confluence. The culture medium was then discarded, and the cells were washed three times with phosphate-buffered saline (PBS solution with a pH of 7.0-7.2). After 72 hours of further culture in commercially available serum-free culture medium, the serum-free culture media from each generation (P3-P30) were collected and combined. Cells not collected were then subjected to further processing. Uncollected cells were digested with a digestive solution (a mixture of 0.25% trypsin and 0.02% EDTA, sterilized by filtration through a 0.22 μm membrane), followed by the addition of cell growth medium to terminate digestion. The cells were centrifuged at 3000 g for 5 minutes at 4°C. The cells were washed three times with PBS and resuspended. They were then sonicated on ice using an ultrasonic disruptor at 400 W for 3 seconds with 6-second intervals for 99 cycles. After the liquid became clear, the supernatant was collected by centrifugation at 17000 g for 30 minutes at 4°C. The supernatants obtained from each generation of human adipose-derived mesenchymal stem cells (P3-P30) were collected and combined. The collected serum-free culture medium and the collected supernatant were mixed at a volume ratio of 1:1, and the solution was filtered through a 0.45 μm filter. The filtrate was collected and concentrated to one-tenth of its original volume. The resulting concentrate was then filtered through a sterile 0.22 μm filter membrane to remove bacteria, thus preparing the human adipose-derived mesenchymal stem cell extract solution.

[0031] The protein concentration of the prepared human adipose-derived mesenchymal stem cell extract solution was determined using the BCA method, and the absorbance at A570 was measured using a microplate reader to obtain the protein concentration. Based on this protein concentration, mannitol (5% of the total weight of the human adipose-derived mesenchymal stem cell extract) and ultrapure water at 50–60°C were added to the final mass of the solution to be lyophilized. The mixture was stirred until the final protein concentration was adjusted to 50.0 ± 0.5 μg / mL. After filtration for sterilization, the solution was lyophilized at -20 to -35°C under a vacuum of 50–200 Pa for 48 hours to obtain the desired lyophilized powder of human adipose-derived mesenchymal stem cell extract. The lyophilized powder was dispensed in 1 mL vials. A lyophilized powder of human adipose-derived mesenchymal stem cell extract was thus prepared.

[0032] Comparative Example 2: Preparation of decellularized biological tissue materials using existing techniques Evenly spread a layer of sterile nonwoven fabric in a clean, sterile tray. After soaking the nonwoven fabric with sterile phosphate buffer, spread the pretreated bovine pericardial tissue on the soaked nonwoven fabric. Gently peel away the obvious adipose tissue layer, avoiding damage to the fibrous layer. Then, with the rough side (fibrous layer) of the biological tissue facing up, gently wipe away the surface fat with a lint-free cloth. Irregular portions and free fibers of the biological tissue were removed, and the tissue was cut into small pieces (15 mm × 15 mm). The tissue was washed three times with physiological saline at room temperature, 3-5 minutes each time. The tissue was then pre-cooled at 4°C for 10 minutes, frozen at -80°C for 2 hours, and thawed in a 37°C water bath for 15 minutes; this process was repeated three times. After thoroughly rinsing the tissue in sterile D-Hanks solution, it was placed in a sealed plastic bag filled with D-Hanks solution and then placed in an ultra-high pressure machine for treatment at 500 MPa and 4°C for 20 minutes. Finally, the tissue was placed in a nuclease solution of 15 μg / mL RNase A and 150 μg / mL DNase I and treated in a constant temperature shaker at 37°C at 100°C. The tissue was rinsed at an oscillation rate of r / min for 48 hours, with the nuclease solution being changed every 24 hours. After rinsing with phosphate buffer for 1 day, the tissue was sealed and stored in phosphate buffer containing antibiotics at 4°C for later use. Table 1 shows the thickness, fracture strength, and DNA content of the biological tissue before and after treatment.

[0033] Comparative Example 3 Following the preparation method provided in Example 1, the 0.1% Triton X-100 decellularization reagent solution was replaced with a 1% Triton X-100 solution, and the other steps were the same as in Example 1, to prepare a lyophilized fiber of a regenerated decellularized lipid derivative.

[0034] Example 2, DNA Residue Test Samples from Example 1 and Comparative Examples 1-3 were mixed with pure water to form solutions. The residual DNA content of the samples was determined by quantitative PCR according to the method for determination of exogenous DNA residue in Chapter 3407 of the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III. The results are shown in Table 1.

[0035] Example 3, VEGF retention rate detection The sample was mixed with pure water to form a solution. A commercially available vascular endothelial growth factor (VEGF) assay kit was used to detect the VEGF concentration in the sample and the VEGF concentration before cell removal. VEGF retention rate = (VEGF concentration of sample before decellularization / VEGF concentration of sample after treatment) × 100% The results are shown in Table 1.

[0036] Example 4, Maximum compressive strength test Take freeze-dried fiber samples to make 1cm 3 A cube was subjected to a constant compressive load using an electronic universal testing machine. The stress-strain curves were recorded to determine the maximum compressive strength of the sample before failure. The results are shown in Table 1.

[0037] Table 1

[0038] Example 5, Facial Filling Test Group 1: Using the lyophilized fiber sample of the regenerated decellularized fat derivative prepared in Example 1, and mixed with physiological saline, a facial filling test was conducted on 50 volunteers in a medical aesthetic clinic.

[0039] Group 2: A facial filling test was conducted on 50 volunteers at a medical aesthetic clinic using commercially available ViiMei recombinant type III collagen.

[0040] Group 3: Using autologous fat as a filler material, a facial filling test was conducted on 50 volunteers at a medical aesthetic clinic.

[0041] The test results for the above three groups are shown in Table 2.

[0042] Table 2

[0043] Example 6, Animal Wound Repair Experiment A full-thickness skin defect (2.0 cm in diameter) model was established in 12 SD rats. Six rats were in the experimental group, filled with the regenerated decellularized fat derivative prepared in Example 1; the other six rats were in the control group, filled with commercially available collagen sponge material. The wound healing rate, inflammatory factor levels, histological scores, and other indicators were observed and measured after 14 days. The results are shown in Table 3 below.

[0044] Table 3

[0045] 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 implemented 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 exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A regenerated decellularized lipid derivative, characterized in that, The raw material for preparation is a vascular matrix component isolated from human adipose tissue, which is obtained by decellularization followed by freeze-drying. The regenerated decellularized adipose-derived fat derivative has a porosity >90% and a pore size of 50-200 μm. Its composition, by weight percentage, includes 60-70% collagen, 15-20% elastin, and 1×10⁻⁶ exosomes. 10 ~5×10 10 Particles / g, DNA residue ≤5 ng / mg, vascular endothelial growth factor content ≥30 ng / g, fibroblast growth factor 2 content ≥15 ng / g.

2. The method for preparing the regenerated decellularized lipid derivative according to claim 1, characterized in that, Including the following steps: S1) Vascular matrix components were isolated from human adipose tissue and amplified to the 3rd to 5th generation under hypoxic conditions to obtain culture products; S2) The culture product was treated with 0.05-0.20% Triton X-100 and a weak alkaline solution to remove cells and cell debris, and a decellularized product was obtained. S3) Freeze-dry the decellularized product to obtain the regenerated decellularized lipid derivative.

3. The preparation method according to claim 2, characterized in that, The amplification under hypoxic conditions refers to culture in DMEM / F12 medium containing 10% fetal bovine serum under conditions of 3% O2 ​​and 5% CO2.

4. The preparation method according to claim 2, characterized in that, The method for separating the vascular matrix components includes: 1) Obtain adipose tissue from a healthy human body, rinse to remove impurities, and cut into small pieces; 2) Add 0.075% type I collagenase solution and digest at 37°C with constant temperature shaking for 30-60 minutes; 3) Filter the tissue suspension using a 200-mesh filter, centrifuge the filtrate at 3000 g for 10-20 minutes at 4°C, and the precipitate is the vascular matrix component.

5. The preparation method according to claim 2, characterized in that, The weak alkaline solution is 0.25% ammonia water.

6. The preparation method according to claim 2, characterized in that, Step S2) The treatment of the culture product includes the following steps: a) Add Triton X-100 to the culture product and shake for 1-2 hours; b) Add an equal volume of 0.25% ammonia solution and shake for 1-2 hours.

7. The preparation method according to claim 2, characterized in that, Step S3) The freeze-drying includes: Pretreatment: Add a protective agent to the decellularized product; Pre-freeze: -80℃, maintain for 2~4 hours; Primary drying: Evacuate to 10 Pa, raise the temperature to -20~-10℃, and maintain until the ice crystals completely sublimate; Desorption and drying: Heat to 25~30℃ and maintain for 6~12 hours.

8. The preparation method according to claim 7, characterized in that, The protective agent is selected from trehalose or glycerol.

9. The preparation method according to claim 7, characterized in that, In the pre-freezing step, the temperature is reduced from room temperature to -40°C at a rate of 1°C / minute, and then reduced to -80°C at a rate of 5°C / minute.

10. The use of the regenerated decellularized fat derivative of claim 1 in the preparation of soft tissue filling materials, wound repair materials, or scar-inhibiting drugs.

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