Composite hydrogel of polycaprolactone / acellular extracellular matrix as well as preparation method and application of composite hydrogel

Abstract

The invention discloses polycaprolactone / acellular extracellular matrix composite hydrogel as well as a preparation method and application thereof, and belongs to the technical field of biological tissue engineering. The amino-modified polycaprolactone nanofiber and the fat acellular matrix are compounded in proportion for the first time, genipin is used as a cross-linking agent of the composite hydrogel, and the hydrogel of an injectable composite material is prepared. The crosslinking degree, the gelation time, the degradation performance, the swelling performance and the mechanical performance of the composite hydrogel with different proportions are studied, and the influence of the composite hydrogel on angiogenesis and fat regeneration in vivo is studied. According to the novel injectable composite hydrogel disclosed by the invention, the 1% PCL / DAT group composite hydrogel has good adipogenesis and vascularization effects, and a new thought is provided for subsequent soft tissue repair and regeneration research.

Description

Technical Field

[0001] The invention belongs to the technical field of biological tissue engineering, and in particular relates to a polycaprolactone / decellularized extracellular matrix composite hydrogel and a preparation method and application thereof. Background Art

[0002] Current research in adipose tissue engineering focuses on how to repair wounds and improve tissue and organ function. Loss of soft tissues, such as skin, fat, and muscle, is a common problem in clinical medicine. Patients experience these losses as a result of tumor resection, trauma, congenital malformations, and natural aging. These tissue losses lead to major functional and aesthetic impairments, making them difficult to treat with conventional methods.

[0003] Hydrogels are scaffold matrices that support tissue regeneration, with three-dimensional (3D) and elastic properties similar to those of soft tissue. Injectable hydrogels are aqueous polymer solutions that gel in situ when deployed in their target environment. They are minimally invasive, can fill irregular cavities, and allow for the co-injection of drugs and biologics. DAT hydrogels have good biocompatibility and injectability. Good cell biocompatibility plays an important role in hydrogels, as it can enhance cell proliferation and differentiation. Constructed hydrogels must possess mechanical properties similar to those of tissues to withstand the physical pressure of surrounding tissues. However, their mechanical strength and resistance to degradation are very low.

[0004] Composites of DAT with decellularized extracellular matrices (SIS) from other tissue sources also suffer from weak mechanical properties and susceptibility to degradation. Furthermore, high-density cross-linking reduces the pore size of the hydrogel network, hindering cell infiltration and resulting in poor regeneration. Creating a porous structure within the hydrogel can facilitate cell infiltration and tissue integration. Summary of the Invention

[0005] In view of the defects of the existing technology, the present invention uses genipin to combine powdered PCL and DAT hydrogel, which can improve the mechanical properties and degradation properties of the composite hydrogel.

[0006] In order to achieve the above objectives, the present invention provides the following technical solutions.

[0007] The invention discloses a composite hydrogel, characterized in that the composite hydrogel comprises polycaprolactone and acellular extracellular matrix.

[0008] Furthermore, the polycaprolactone in the composite hydrogel is amino-modified polycaprolactone.

[0009] Furthermore, the composite hydrogel consists of 1% PCL / DAT.

[0010] Furthermore, the composite hydrogel is in an injectable form.

[0011] The present invention discloses an application of any of the above-mentioned composite hydrogels in preparing a vascularized tissue engineering adipose tissue scaffold.

[0012] The present invention discloses an application of any of the above-mentioned composite hydrogels in the preparation of soft tissue repair and regeneration materials.

[0013] The present invention discloses a method for preparing the composite hydrogel as described above, characterized in that the preparation method specifically comprises the following steps: Step 1 DAT preparation: (1) Cut the pig fat and skin off, then chop into small pieces, wash with distilled water to remove impurities, rinse with PBS three times, add sufficient distilled water, and soak in a constant temperature shaker at 37°C for 20 minutes; (2) Soak the sheared adipose tissue after treatment in (1) in 1 mol / L sodium chloride solution for 3 h, then soak it in PBS and shake it in a constant temperature shaker at 37°C for 1 h; (3) Take out the tissue treated in (2), rinse it with PBS three times, and cut it into pieces with a size of 0.4~0.8cm. 3 The blocks were transferred into plastic bottles and placed in a -80°C ultra-low temperature freezer overnight, and then placed in a 37°C water bath to melt. This process was repeated 4 to 5 times. (4) Add an appropriate amount of distilled water to the adipose tissue treated in (3) and homogenize and crush it at a speed of 10,000 rpm for 5 minutes at room temperature; centrifuge the homogenized liquid at 12,000 rpm for 5 minutes, collect the white flocculent matter at the bottom after centrifugation, remove the upper layer, and repeat the homogenization and centrifugation of the remaining white fat in the middle layer; (5) Place the white precipitate collected in (4) in a 1% Trion-x100 solution and shake at 37°C for 1 hour; rinse three times with distilled water, then place the white flocculent in isopropanol and shake at 37°C for 48 hours; (6) Add to 1 mol / L sodium chloride solution and shake at 37°C overnight; centrifuge the shaken flocs at 1000g, 4°C, 5 min, rinse three times with distilled water, freeze-dry, and grind into powder using a low-temperature wall-breaking grinder to obtain DAT; sterilize the powdered DAT and store in a -80°C refrigerator; Step 2 Preparation of PCL powder: (1) PCL was dissolved in hexafluoroisopropanol (HFIP) and stirred at room temperature overnight. The electrospinning injection rate was 2.5 mL / h. A voltage of 16 kV was applied to the metal needle, and the distance between the needle tip and the ground was 12 cm. (2) The PCL prepared in (1) was collected, sheared, crushed, and filtered through a 100-mesh filter. The PCL was placed in a 0.43 mol / L 1,6-hexanediamine methanol solution and soaked for 8 h. The PCL was then rinsed with PBS three times and freeze-dried to obtain amino-modified PCL powder. Step 3 Preparation of composite hydrogel: (1) Dissolve pepsin in 0.01 mol / mL dilute hydrochloric acid to prepare a 1 mg / mL pepsin hydrochloride solution, and filter with a 0.22 μm filter; add the sterilized DAT powder prepared in step 1 at 4°C and stir thoroughly for 48 hours until no powdery substance remains; (2) The solution prepared in (1) was adjusted to pH 7.4 with 0.1 mol / mL sodium hydroxide, and finally one-ninth of the total volume of 10× PBS was added and stored at 4°C; (3) The DAT hydrogel prepared in (2) was mixed with 1% of the PCL powder prepared in step 2 and cross-linked with 0.2% genipin (w / v) to prepare a composite hydrogel.

[0014] Furthermore, the sodium chloride solution in step 1 (6) contains 100 ug / mL DNase and 100 ug / mL RNase.

[0015] Furthermore, in step 2 (1), the mass-to-volume ratio of PCL to HFIP is 1:10.

[0016] Furthermore, the concentration of the DAT hydrogel in step three (3) is 8 mg / mL.

[0017] Compared with the prior art, the present invention has the following beneficial effects.

[0018] The present invention innovatively combines the high molecular weight synthetic polymer with the natural decellularized extracellular matrix material in this experiment, and for the first time proposes combining the two substances DAT / PCL to prepare an injectable composite hydrogel, forming a synergistic relationship, enhancing the physical properties and biological activity of the scaffold, thereby eliminating their respective disadvantages and achieving the characteristics of compensating for each other's shortcomings.

[0019] In the composite hydrogel disclosed in the present invention, PCL is creatively crushed and then aminolyzed. Studies have found that the aminolyzed PCL has the effect of promoting cell adhesion and proliferation, thereby promoting angiogenesis.

[0020] The composite hydrogel disclosed in the present invention is injected, which is simple and minimally invasive to operate, has wide clinical applications, and has high safety and practicality.

[0021] After the composite hydrogel prepared by cross-linking the powdered PCL and DAT hydrogel disclosed in the present invention was implanted subcutaneously in rats, macrophages infiltrated first in the early stage, and stem cells did not enter the composite hydrogel. The expression of stem cells increased over time, indicating that the composite hydrogel recruited stem cells and was beneficial for stem cells to promote tissue regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The figure shows the flow chart for preparing composite hydrogels. A is the flow chart for preparing DAT, and B is the flow chart for preparing PCL powder.

[0023] Figure 2 The results of DAPI and Oil Red O staining of fresh materials after decellularization. Figures A and B show the DAPI staining results of adipose tissue and DAT, respectively, and C and D show the Oil Red O staining results of adipose tissue and DAT, respectively.

[0024] Figure 3 Figure 1 shows the physicochemical properties of the composite hydrogels. A shows the DNA quantitative analysis results; B shows the agarose electrophoresis; C shows a gel image (left: PCL-crosslinked DAT hydrogel; right: PCL-crosslinked DAT hydrogel with amino groups introduced); D shows the gelation time graph and gelation time statistical analysis; E shows the in vitro degradation curve of the hydrogels; F shows the hydrogel swelling ratio; and G shows the amino content of the hydrogels.

[0025] Figure 4 Mechanical properties of composite hydrogel.

[0026] Figure 5 Drawings for animals.

[0027] Figure 6 Statistical results of endothelial cell proliferation detection.

[0028] Figure 7 AO / PI staining of endothelial cells.

[0029] Figure 8 The results are HE staining.

[0030] Figure 9 Alizarin red staining. A is the DAT group, B is the 0.5% PCL / DAT group, C is the 1% PCL / DAT group, and D is the 2% PCL / DAT group.

[0031] Figure 10 Oil Red O staining pictures and statistical results.

[0032] Figure 11 These are the images and statistical results of immunohistochemistry PerilipinA.

[0033] Figure 12These are the images and statistical results of immunohistochemistry CD31.

[0034] Figure 13 These are the images and statistical results of immunohistochemistry CD105.

[0035] Figure 14 Comparison of PCL preparation results. A shows the increased hydrophilicity of PCL nanofibers after the introduction of amino groups; B shows the microstructure of PCL / DAT hydrogels at different ratios, with arrows indicating PCL nanofibers; and C shows that 1% PCL / DAT significantly promotes the formation of endothelial cell tubular structures. DETAILED DESCRIPTION

[0036] The following examples will help to understand the present invention, but these examples are only for illustration of the present invention and should not be understood as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. The present invention is not limited to these contents. The operating methods in the examples are all conventional operating methods in the art.

[0037] Example 1 Preparation of the composite hydrogel of the present invention.

[0038] 1. Experimental materials.

[0039] 1. Materials: fresh porcine fat, polycaprolactone (MW = 80,000 Da).

[0040] 2. Main reagents: saturated Oil Red O stock solution, hexafluoroisopropanol, xylene, DAPI, anhydrous ethanol, 1,6-hexanediamine.

[0041] 2. Experimental methods

[0042] 1. DAT preparation process ( Figure 1 A).

[0043] (1) Cut the skin off the purchased pig fat and chop it into small pieces. Wash away impurities with distilled water. Rinse with PBS three times and then add enough distilled water. Place in a constant temperature shaker at 37°C and soak for 20 minutes.

[0044] (2) Soak the sheared adipose tissue in 1 mol / L sodium chloride solution for 3 h.

[0045] (3) Soak in PBS and shake in a constant temperature shaker at 37°C for 1 hour.

[0046] (4) After removing the tissue, rinse it with PBS three times and cut the fat tissue into a size of 0.4~0.8cm 3 piece.

[0047] (5) Place a small piece of fat tissue into a plastic bottle, place it in a -80°C ultra-low temperature freezer overnight, and then place it in a 37°C water bath to melt. Repeat this process 4 to 5 times.

[0048] (6) Add an appropriate amount of distilled water to the adipose tissue and homogenize it at 10,000 rpm for 5 minutes at room temperature.

[0049] (7) Centrifuge the homogenized liquid (12000 rpm, 5 min), collect the white flocculent matter at the bottom, remove the upper layer, and repeat the homogenization and centrifugation of the remaining white fat in the middle layer.

[0050] (8) Place the collected white precipitate in 1% Trion-x100 solution and shake at 37°C for 1 hour.

[0051] (9) After rinsing three times with distilled water, the white flocs were placed in isopropanol and shaken at 37°C for 48 h.

[0052] (10) Add 1 mol / L (100 μg / mL DNA enzyme + 100 μg / mL RNA enzyme) sodium chloride solution and shake at 37°C overnight.

[0053] (11) The flocculent material after shaking was centrifuged at 1000 g for 5 min at 4°C, rinsed three times with distilled water, freeze-dried, and ground into powder using a low-temperature wall-breaking grinder.

[0054] (12) Sterilize the powder and store it in a -80℃ refrigerator. Observe the gel under a scanning electron microscope.

[0055] 2. Preparation of PCL powder: PCL was dissolved in hexafluoroisopropanol (HFIP) at a PCL / HFIP mass-to-volume ratio of 1:10. The mixture was stirred at room temperature overnight. The electrospinning rate was 2.5 mL / h, and a voltage of 16 kV was applied to the metal needle. The distance between the needle tip and the ground was 12 cm. The PCL was collected, sheared, and crushed, then filtered through a 100-mesh filter. The PCL was then placed in a 0.43 mol / L 1,6-hexanediamine methanol solution and soaked for 8 hours. The mixture was then rinsed three times with PBS and freeze-dried. After surface gold coating, the pores of the PCL were observed using a scanning electron microscope (SEM). Figure 1 B).

[0056] 3. Preparation of composite hydrogel: Dissolve pepsin in 0.01 mol / mL dilute hydrochloric acid to prepare a 1 mg / mL pepsin hydrochloride solution, and filter with a 0.22 μm filter. Add sterilized DAT powder at 4°C and stir thoroughly for 48 hours until there is no powdery substance. Adjust the pH of the solution to 7.4 with 0.1 mol / mL sodium hydroxide, and finally add one-ninth of the total volume of 10×PBS and store at 4°C. The concentration of DAT hydrogel is 8 mg / mL. The DAT hydrogel is mixed with PCL powder of different concentrations, cross-linked with 0.2% concentration of genipin, and the injectable composite hydrogel of the present invention is prepared.

[0057] Example 2 Physicochemical properties of the injectable composite hydrogel of the present invention.

[0058] 1. Experimental methods

[0059] (I) Histological examination: To evaluate the results of the decellularization method, fresh porcine adipose tissue and DAT were embedded and frozen, and then the sections were stained with DAPI and Oil Red O. DAPI staining steps: (1) Soak the sections in distilled water for 2 minutes. (2) Soak in 4% paraformaldehyde for 15 minutes and then wash with distilled water. (3) Overlay the DAPI stain solution on the tissue and stain in the dark for 5 minutes. (4) Rinse with PBS three times and then mount the sections. (5) Observe under a fluorescence microscope.

[0060] (2) Detection of residual DNA.

[0061] 25 mg of adipose tissue and DAT were ground into powder using liquid nitrogen and added to 1.5 mL EP tubes. Genomic DNA was then extracted from adipose tissue and DAT using an animal tissue genomic DNA extraction kit (Solaibao, China). DNA content was measured.

[0062] (3) Agarose electrophoresis. To prepare agarose hydrogel: Place 0.7 g of agarose in a conical flask, add 70 mL of electrophoresis buffer, shake well, and microwave three times until the agarose dissolves. Pour the heated agarose hydrogel into the inner glass tank and let it stand at room temperature until the gel solidifies. Pull out the comb vertically, add the gel and inner tank to the electrophoresis tank, and add electrophoresis buffer until the gel is submerged. Add the DNA sample and buffer. Charge the electrophoresis; run at 90 V; terminate the voltage; turn off the power, and observe and photograph the gel using a gel imager.

[0063] (IV) Preparation of composite hydrogels. Dissolve pepsin in 0.01 mol / mL dilute hydrochloric acid to prepare a 1 mg / mL pepsin hydrochloride solution, which was then filtered through a 0.22 μm filter. Add sterilized DAT powder at 4°C and stir thoroughly for 48 hours until no powdery material remains. Adjust the pH of the solution to 7.4 with 0.1 mol / mL sodium hydroxide, and finally add one-ninth the total volume of 10× PBS and store at 4°C. The concentration of the DAT hydrogel was 8 mg / mL. The DAT hydrogel was mixed with different concentrations of PCL powder according to the ratios described in Table 1 and cross-linked with 0.2% genipin.

[0064] Table 1 Composite hydrogel ratio .

[0065] (V) Gel time and degradation analysis.

[0066] Gel time: Place each group of composite hydrogels in a 37°C environment and observe the gel flow by tilting the reagent bottle every half a minute.

[0067] Degradation: After gelling, the same volume of hydrogel in each group was washed with PBS and weighed W1. After weighing, they were placed in PBS buffer and then placed in a 37°C environment. Samples were taken out at 1, 3, 7, 14, and 28 days, and the surface moisture was wiped off with filter paper. The gel was weighed W2.

[0068] , where W1 is the initial measurement of the wet weight of the hydrogel, and W2 is the wet weight measured at different time periods.

[0069] (VI) Swelling rate test: Place the three samples in separate 15 mL centrifuge tubes and add an appropriate amount of PBS buffer. After complete swelling, remove the gels and remove the water with filter paper. Weigh M1. Then, cool and dry the hydrogels and weigh them as M0.

[0070] , where M1 is the weight of the hydrogel measured initially, and M0 is the weight of the hydrogel after cold drying.

[0071] (VII) Determination of amino group cross-linking. The cross-linking degree of the hydrogel was analyzed using the ninhydrin colorimetric method. 1 mL of hydrogel was mixed with 2 mL of deionized water and 1 mL of a 2% ninhydrin solution. The mixture was preheated at 100°C for 10 minutes and cooled to room temperature to terminate the reaction. Furthermore, free amino groups were measured spectrophotometrically (Multiscan FC, Thermo Scientific) at 570 nm. Various concentrations of glycine were prepared for the standard curve.

[0072] , where (NH2) a is the molar fraction of free amino groups in the hydrogel, (NH2) b is the mole fraction of free amino groups in the genipin cross-linked hydrogel.

[0073] (8) Mechanical Properties Testing. The mechanical properties of the hydrogels were measured using a materials testing machine. 1.5 mL of hydrogel was cast into a cylindrical shape in a 24-well plate, and the initial height and diameter were recorded. A press was used to apply pressure to the hydrogel, slowly compressing it at a rate of 6 mm / s. The maximum elastic modulus was determined by calculating the slope of the stress-strain curve.

[0074] 2. Experimental results.

[0075] (I) Decellularization results. The cell nuclei of the fresh material group were blue. After the adipose tissue was decellularized, DAPI staining showed no cell nuclei, indicating that the tissue was very clean after decellularization. Oil Red O staining was used to observe the degreasing of the oily material. It was found that the fresh fat droplets were bright red, while the decellularized group had no obvious red color, indicating that the fat droplets had been completely shed. Figure 2 , scale bar = 50 μm).

[0076] (II) DNA quantitative analysis results. DNA detection kits were used to quantitatively detect fresh adipose tissue (N-DAT) and DAT. The DNA content of the fresh adipose tissue group was 1928.64±29.85ng / mg, and the DNA content of the DAT group was 39.2±3.73ng / mg ( Figure 3 A, Data are expressed as mean ± standard deviation (n = 3); comparisons among groups were performed using one-way analysis of variance; *** P <0.001, compared with the N-DAT group).

[0077] (III) Agarose electrophoresis. By comparing the electrophoresis results of adipose tissue (N-DAT) and DAT, it can be found that the DNA in adipose tissue contains complex components, while the amount of DNA in DAT is small, indicating that DAT is cleanly washed ( Figure 3 B).

[0078] (IV) Composite hydrogel cross-linking results. The gel image results show that PCL without amino group introduction is more distributed in the lower layer of DAT hydrogel. In the right image, PCL is evenly distributed, indicating that amino group introduction is successful in PCL, and NH2-PCL is cross-linked with DAT hydrogel ( Figure 3 C).

[0079] (V) Gel time. This experiment compared the differences in gel time among the groups. The gel time of the DAT group was 990s, the gel time of the 0.5% PCL / DAT group was 793.3s, the gel time of the 1% PCL / DAT group was 551.6s, and the gel time of the 2% PCL / DAT group was 276.6s. The experimental results suggest that as the amount of PCL increases, the gel time decreases ( Figure 3 D, **** P <0.0001 compared with DAT group, #### P <0.001 vs. 2% PCL / DAT group).

[0080] (VI) In vitro degradation. During the degradation period, the degradation rate of the 2% PCL / DAT group was significantly lower than that of the other three groups. The degradation rate in the fourth week was less than 20%, while the degradation rate of DAT+G was greater than 50%. This shows that the degradation rate of the hydrogel was significantly reduced after the addition of PCL. Figure 3 E).

[0081] (VII) Swelling rate determination. The equilibrium swelling rate of the hydrogel after swelling to equilibrium ranged from 773% to 1363%. The swelling rate of the DAT group was 1345.69%, the swelling rate of the 0.5% PCL / DAT group was 980.433%, the swelling rate of the 1% PCL / DAT group was 880.527%, and the swelling rate of the 2% PCL / DAT group was 765.217%. The swelling rate of the DAT group was much greater than that of the other groups. Figure 3 F, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 vs. DAT group; ## P <0.01, #### P <0.001 compared with 0.5% PCL / DAT group, && P < 0.01 vs. 1% PCL / DAT group, n = 3).

[0082] (8) Determination of cross-linking degree. Ninhydrin reacts with amino groups under heating conditions to form a purple compound proportional to the amino acid concentration. The amino acid content can be determined by measuring the optical density at 570 nm. The cross-linking degree of the DAT group was 79.55%, the cross-linking degree of the 0.5% PCL / DAT group was 78.82%, the cross-linking degree of the 1% PCL / DAT group was 75.64%, and the cross-linking degree of the 2% PCL / DAT group was 72.93%. Figure 3 G).

[0083] (IX) Mechanical properties test results. The mechanical properties test results showed that the elastic modulus of the DAT group was 7.3KPa, the elastic modulus of the 0.5% PCL / DAT group was 13.0KPa, the elastic modulus of the 1% PCL / DAT group was 15.2KPa, and the elastic modulus of the 2% PCL / DAT group was 18.0KPa ( Figure 4 , ** P <0.01, *** P <0.001, **** P <0.0001 vs. DAT group; ## P <0.01, compared with the 0.5% PCL / DAT group, n=3).

[0084] 3. Experimental conclusions.

[0085] After decellularization, adipose tissue forms white particles called DAT. DNA quantification revealed a DNA content of 39.2 ± 3.73 ng / mg in the DAT group. DAPI staining revealed the absence of cell nuclei, and Oil Red O staining revealed no residual lipid droplets, confirming complete decellularization of the DAT.

[0086] Genipin was used to cross-link DAT and PCL to form composite hydrogels. The gelation time of each group was observed. The 2% PCL / DAT group had the fastest gelation time, while the DAT group had the shortest gelation time, indicating that the addition of PCL powder facilitated rapid gelation. In degradation rate testing, the degradation rate of DAT was much greater than that of the 2% PCL / DAT group, suggesting that the addition of PCL reduced the degradation rate of the hydrogel. In swelling rate measurements, the swelling rate decreased with the addition of PCL, likely due to the numerous pores in the electrospun PCL, which reduced the space available for water retention.

[0087] Ninhydrin reacts with free amino acids to form a blue compound. After genipin crosslinks with the hydrogel, the color of the gel also changes. Therefore, when conducting this experiment, a genipin solution is added as a control to remove the color effect of genipin crosslinking in the calculations. PCL was fully dissolved in hexafluoroisopropanol, and then PCL fragments were prepared using an electrospinning device. After pulverization, amino groups were introduced. The degree of crosslinking was measured using the ninhydrin method. The crosslinking degree of the DAT group was 79.55%, the 0.5% PCL / DAT group had a crosslinking degree of 78.82%, the 1% PCL / DAT group had a crosslinking degree of 75.64%, and the 2% PCL / DAT group had a crosslinking degree of 72.93%. The crosslinking degree in each group was greater than 70%.

[0088] In the mechanical properties test, the results showed that the elastic modulus of the DAT group was 7.3KPa, the elastic modulus of the 0.5% PCL / DAT group was 13.0KPa, the elastic modulus of the 1% PCL / DAT group was 15.2KPa, and the elastic modulus of the 2% PCL / DAT group was 18.0KPa, while the elastic modulus of human subcutaneous fat was 10.90±4.80KPa. The elastic moduli of the first three groups were close to those of subcutaneous fat, and the elastic modulus of the 2% PCL / DAT group was greater than that of subcutaneous fat.

[0089] Example 3 In vitro and in vivo studies of composite hydrogels.

[0090] 1. Experimental materials.

[0091] 1 Experimental materials: human adipose-derived stem cells (hASCs) and human umbilical vein endothelial cells (HUVECs).

[0092] 2 Experimental Animals: Male Wistar rats, 6 weeks old, weighing 150–180 g, 8 rats per group. The animal experiments were approved by the Ethics Review Committee of China Medical University (Ethics Number: CMU2022069).

[0093] 3 Experimental reagents: HE staining solution, Alizarin red staining solution, 4% paraformaldehyde, DAB color developer.

[0094] 2. Experimental methods

[0095] 1. Cell Culture: Immediately place cells from liquid nitrogen in a 37°C water bath. Thaw and quickly remove cells. Add the same volume of culture medium. Centrifuge at 1000 rpm at 4°C for 5 minutes. Discard the supernatant and culture with complete culture medium (10% FBS + 1% double-antibody) at 37°C in 5% CO2. Change the medium every 3 days. When the concentration reaches 80%-90%, passage the cells. Select passages 4-8 for subsequent experiments.

[0096] 2. Cell Proliferation Assay: Cell proliferation was measured using CCK-8 reagent. Cells were seeded in 96-well plates at a density of 2000 cells / well. After cell attachment, the extract was replaced with the culture medium. After 1, 3, and 5 days of culture, 10 μL of CCK-8 solution was added to each well. The cells were incubated at 37°C in the dark for 2 hours. The absorbance at 450 nm was measured using a microplate reader. A control group and a blank group were established. The control group received standard culture medium and cells, while the blank group received standard culture medium.

[0097] 3. Cytotoxicity Assay: AO (Acridine Orange) is a membrane-permeable fluorescent dye that causes cell nuclei to appear yellow-green; PI (Propidium Iodide) is a DNA-binding dye that causes dead cell nuclei to appear red. Add the hydrogel to prepare an extract, seed endothelial cells, and culture for 1, 3, and 5 days. Wash the cells three times with PBS and then add an appropriate volume of staining solution and incubate at 37°C for 20 minutes. Aspirate the staining solution, wash the culture plate twice in culture medium, and photograph using an inverted fluorescence microscope.

[0098] 5. Animal Experiments. Four groups of hydrogels were injected subcutaneously into rats, with each rat receiving four injection sites: the DAT group, the 0.5% PCL / DAT group, the 1% PCL / DAT group, and the 2% PCL / DAT group. The rats were anesthetized using an anesthesia machine and the composite hydrogel was injected into the back using a 1 mL syringe, with each rat receiving four injection sites. The rats were euthanized on days 7, 14, and 28, respectively, and samples were collected and photographed. Figure 5 , scale bar = 1 cm).

[0099] 6. Frozen Sections: Place the specimen in 4% paraformaldehyde solution at 4°C overnight. Then, place the specimen in 20% sucrose overnight and then in 30% sucrose overnight. After precipitation, dry the specimen with filter paper and freeze in liquid nitrogen. Once the tissue turns bleached, store in a -80°C freezer. After embedding in OCT, slice the specimen using a cryostat.

[0100] 7. Hematoxylin-eosin (HE) staining: (1) Rinse the frozen sections with distilled water. (2) Fix the specimens with 4% paraformaldehyde for 10 minutes. (3) Soak in distilled water for 2 minutes. (4) Dilute the hematoxylin fivefold with distilled water and stain for 20 minutes. (5) Soak in distilled water for 1 minute. (6) Rinse with 1% hydrochloric acid ethanol solution for 3 seconds. (7) After blueing, rinse with tap water for 20 minutes. (8) Stain with 1% eosin solution for 3 minutes. (9) Wash with distilled water. (10) Dehydrate with graded alcohol and clear with xylene. (12) Mount with neutral gum.

[0101] 8. Oil Red O staining: (1) Fix sections with 4% paraformaldehyde for 10 minutes. (2) Rinse with distilled water for 1 minute. (3) Immerse in 60% isopropanol for 3-5 seconds. (4) Stain with Oil Red O for 20 minutes. (5) Differentiate with 60% isopropanol until the stroma is clear. (6) Rinse with distilled water. (7) Counterstain with hematoxylin for 5 minutes. (8) Wash with distilled water and mount with glycerol gelatin.

[0102] 9. Alizarin red staining: (1) Soak sections in PBS twice, each for 5 minutes. (2) Fix in 95% alcohol for 10 minutes. (3) Wash three times in distilled water, each for 5 minutes. (4) Stain with 0.2% alizarin red for 30 minutes. (5) Dehydrate in graded alcohol and clear with xylene. (6) Mount with neutral gum.

[0103] 10. Immunohistochemistry. (1) Rinse the frozen tissue with distilled water for 1 minute. (2) Immerse in 3% hydrogen peroxide solution at room temperature, incubate in the dark for 5 minutes, and rinse three times with PBS, each for 5 minutes. (3) Serum blocking: Add BSA to the tissue until it is completely immersed, and block at 37°C for 30 minutes. (4) Add primary antibody: Discard the blocking solution, no need to wash with water, add PBS solution to the control group, and add primary antibody to the experimental group, and incubate at 4°C overnight. (5) Add secondary antibody: Wash with PBS three times, each for 5 minutes; shake dry slightly, and incubate the secondary antibody at 37°C for 30 minutes. (6) DAB color development: Wash with PBS three times, each for 5 minutes, shake dry slightly, and add DAB color development solution to the tissue. When the tissue shows a brown-yellow positive reaction, rinse with distilled water to complete the color development. (7) Counterstain the nucleus: Counterstain the nucleus with hematoxylin for about 5 minutes, differentiate with 1% hydrochloric acid and ethanol, wash away the excess dye in the nucleus until the stain is clear and bright, and rinse with tap water for 20 minutes. (8) Dehydrate with alcohol gradient, clear with xylene, and then seal with neutral gum.

[0104] 11. Statistical Analysis. One-way analysis of variance was used to assess the significance of differences between groups. Statistical analyses were performed using GraphPad Prism 7.0. P < 0.05 was considered statistically significant.

[0105] 3. Experimental results.

[0106] 1. Composite hydrogels promote endothelial cell proliferation. To verify the effect of composite hydrogels on cell proliferation, we treated endothelial cells with different groups of hydrogel extracts. CCK-8 results showed that cell viability was greater than 80% on days 1, 3, and 5, indicating that the composite hydrogels were non-toxic to the cells. On day 3, the 2% PCL / DAT group had the best proliferation-promoting effect. On day 5, the 1% PCL / DAT and 2% PCL / DAT groups had the most significant ability to promote endothelial cell proliferation ( Figure 6 , * P <0.05, **** P <0.0001 vs. DAT group; ## P <0.01, #### P <0.001 vs. 0.5% PCL / DAT group, && P <0.01 vs. 1% PCL / DAT group, n = 5).

[0107] 2. The composite hydrogel is non-toxic. After plating the endothelial cells, the culture medium was replaced with the extract. AO / PI staining was performed on day 1, day 3, and day 5. Green fluorescence indicated live cells, while red indicated dead cells. Cytotoxicity results showed good cell activity, demonstrating that the hydrogels in each group were non-toxic to the cells ( Figure 7 , scale bar = 100 μm).

[0108] 4. In vivo histological staining of the composite hydrogel. HE staining results showed cell infiltration within the hydrogel. The DAT group had fewer cells at each time period, while the other groups had more cells than the DAT group, indicating that the addition of PCL promoted cell infiltration. Furthermore, HE staining results clearly showed angiogenesis, with the 1% PCL / DAT group having the most blood vessels ( Figure 8 , scale bar = 50 μm).

[0109] Alizarin red staining results showed that on day 28, a small amount of calcium deposition was observed in the 2% PCL / DAT group, while no calcium deposition was observed in the other three groups. These results indicate that the 2% PCL / DAT group will undergo calcification in the later stages and is not suitable for soft tissue repair. Figure 9 , scale bar = 50 μm).

[0110] 5. Composite hydrogel promotes adipogenesis in vivo. After the composite hydrogel was implanted subcutaneously in rats, samples were collected on the 7th, 14th, and 28th day. The samples were frozen and sectioned, and then stained with Oil Red O. The results showed that no obvious lipid droplets were observed on the 7th day. On the 14th day, lipid droplets were clearly observed in the 0.5% PCL / DAT group, the 1% PCL / DAT group, and the 2% PCL / DAT group. The 1% PCL / DAT group had the best lipid formation effect. On the 28th day, lipid droplets were observed in all groups, and the 1% PCL / DAT group had the best lipid formation effect ( Figure 10 , * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 vs. DAT group; # P <0.05, ## P <0.01, #### P <0.001 vs. 0.5% PCL / DAT group, & P <0.05 vs. 1% PCL / DAT group. NC, negative control. Scale bar = 50 μm, n = 5).

[0111] PerilipinA marks lipid droplet coating proteins, indicating the formation of lipid droplets in the material. Brown-yellow represents positive staining results. The results showed that the 1% PCL / DAT combination had the best lipidation effect. The results of Oil Red O and immunohistochemistry PerilipinA were consistent, and the 1% PCL / DAT combination had a stronger lipidation effect than the other three groups ( Figure 11 , *** P <0.001, **** P <0.0001 vs. DAT group; # P <0.05, ## P <0.01 vs. 0.5% PCL / DAT group. NC, negative control. Scale bar = 50 μm, n = 5).

[0112] 6. The composite hydrogel promotes angiogenesis in vivo. CD31 is a marker for endothelial cells that can detect the presence of blood vessels, with brown-yellow staining indicating positive results. No significant angiogenesis was observed on day 7 after implantation, but significant angiogenesis was observed on days 14 and 28. The DAT group had the lowest CD31 expression, while the 1% PCL / DAT and 2% PCL / DAT groups had the highest CD31-positive cells ( Figure 12 , * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 vs. DAT group; # P <0.05, compared with the 0.5% PCL / DAT group. NC, negative control. Scale bar = 50 μm, n = 5).

[0113] 8. Effect of composite hydrogel on stem cell recruitment. CD105 is a common surface marker of mesenchymal cells, and brown-yellow indicates positive staining results. No positive indicators were observed on day 7. On day 14, the number of CD105-positive cells in the 2% PCL / DAT group and the 1% PCL / DAT group was significantly higher than that in the DAT group. On day 28, the number of positive cells in the 2% PCL / DAT group was higher than that in the other groups ( Figure 13 , * P <0.05 vs. DAT+G group. NC, negative control. Scale bar = 50 μm, n = 5).

[0114] 3. Experimental conclusions.

[0115] DAT hydrogel contains many proteins and growth factors, which can promote fat regeneration and angiogenesis. DAT not only has good biocompatibility, but can also be used as a drug carrier. The porosity generated by the polymer affects the diffusion of oxygen and nutrients. Porosity is the percentage of void space in the material, which represents a part of the total volume. Porosity affects contact guidance and cell directional differentiation during cell migration. Better porosity can induce cell aggregation and promote proliferation. PCL contains a large number of pores after electrospinning, which can promote cell infiltration and adhesion. PCL after aminolysis also has the effect of promoting cell adhesion and cell proliferation, which is beneficial to the formation of blood vessels ( Figure 14 ). In addition, PCL acts as a three-dimensional scaffold in the hydrogel, providing mechanical support. After cross-linking PCL with DAT hydrogel, the elastic modulus increases, and the elastic modulus of 0.5% PCL / DAT and 1% PCL / DAT approaches that of adipose tissue. The elastic modulus of the 2% PCL / DAT group is 18.0 kPa, which is greater than the elastic modulus of human subcutaneous fat of 10.90 ± 4.80 kPa. In this experiment, the lipogenic effect of the 2% PCL / DAT group was not as good as that of the 1% PCL / DAT group. The reason may be that the excessive PCL content leads to an excessive elastic modulus. Therefore, Alizarin Red staining results showed that calcium deposition occurred in the 2% PCL / DAT group after in vivo transplantation.

[0116] Biomaterials can be designed to recruit stem cells and coordinate their behavior and function to restore or replace damaged or diseased tissues. Experiments have shown that the CD105 positive expression rate in the 2% PCL / DAT group was higher than that in other groups. The reason may be that the stem cells were inoculated on the polymeric three-dimensional scaffold, and the stem cells increased due to the increase in their three-dimensional scaffold. After the composite hydrogel was implanted subcutaneously in rats, the expression of stem cells increased over time, indicating that the composite hydrogel recruited stem cells and helped stem cells promote tissue regeneration. The present invention prepared a new injectable composite hydrogel. The 1% PCL / DAT group composite hydrogel has good adipogenesis and angiogenesis effects, which provides a new idea for the subsequent soft tissue repair and regeneration research.

[0117] In summary, the present invention combines a decellularized extracellular matrix hydrogel with a high molecular weight polymer after aminolysis for the first time, obtaining a hydrogel with good promotion of fat regeneration and angiogenesis. Further exploration of the gel time, degradation performance, swelling rate, mechanical properties, and other characteristics of the composite hydrogels with different ratios revealed that the 1% PCL / DAT group had the best effect in terms of fat regeneration and angiogenesis, and the 1% PCL / DAT group had far better fat regeneration and angiogenesis effects than the DAT group. Moreover, this composite hydrogel is a temperature-sensitive hydrogel and can be made into a hydrogel preform and transported in a low-temperature environment to meet the clinical needs of drug delivery or repairing soft tissue injuries.

[0118] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A composite hydrogel, characterized in that The composite hydrogel comprises polycaprolactone and decellularized extracellular matrix.

2. The composite hydrogel according to claim 1, characterized in that The polycaprolactone in the composite hydrogel is amino-modified polycaprolactone nanofiber.

3. The composite hydrogel according to claim 1, characterized in that The composite hydrogel consists of 1% PCL / DAT.

4. The composite hydrogel according to claim 1, characterized in that The dosage form of the composite hydrogel is injection type.

5. Use of the composite hydrogel according to any one of claims 1 to 4 in preparing a vascularized tissue engineering adipose tissue scaffold.

6. Use of the composite hydrogel according to any one of claims 1 to 5 in preparing soft tissue repair and regeneration materials.

7. A method for preparing the composite hydrogel according to claim 1, characterized in that: The preparation method specifically comprises the following steps: Step 1 DAT preparation: (1) Cut the pig fat and skin off, then chop into small pieces, wash with distilled water to remove impurities, rinse with PBS three times, add sufficient distilled water, and soak in a constant temperature shaker at 37°C for 20 minutes; (2) Soak the sheared adipose tissue after treatment in (1) in 1 mol / L sodium chloride solution for 3 h, then soak it in PBS and shake it in a constant temperature shaker at 37°C for 1 h; (3) Take out the tissue treated in (2), rinse it with PBS three times, and cut it into pieces with a size of 0.4~0.8cm. 3 The blocks were transferred into plastic bottles and placed in a -80°C ultra-low temperature freezer overnight, and then placed in a 37°C water bath to melt. This process was repeated 4 to 5 times. (4) Add an appropriate amount of distilled water to the adipose tissue treated in (3) and homogenize and crush it at a speed of 10,000 rpm for 5 minutes at room temperature; centrifuge the homogenized liquid at 12,000 rpm for 5 minutes, collect the white flocculent matter at the bottom after centrifugation, remove the upper layer, and repeat the homogenization and centrifugation of the remaining white fat in the middle layer; (5) Place the white precipitate collected in (4) in a 1% Trion-x100 solution and shake at 37°C for 1 hour; rinse three times with distilled water, then place the white flocculent in isopropanol and shake at 37°C for 48 hours; (6) Add to 1 mol / L sodium chloride solution and shake at 37°C overnight; centrifuge the shaken flocs at 1000g, 4°C, 5 min, rinse three times with distilled water, freeze-dry, and grind into powder using a low-temperature wall-breaking grinder to obtain DAT; sterilize the powdered DAT and store in a -80°C refrigerator; Step 2 Preparation of PCL powder: (1) PCL was dissolved in hexafluoroisopropanol (HFIP) and stirred at room temperature overnight. The electrospinning injection rate was 2.5 mL / h. A voltage of 16 kV was applied to the metal needle, and the distance between the needle tip and the ground was 12 cm. (2) The PCL prepared in (1) was collected, sheared, crushed, and filtered through a 100-mesh filter. The PCL was placed in a 0.43 mol / L 1,6-hexanediamine methanol solution and soaked for 8 h. The PCL was then rinsed with PBS three times and freeze-dried to obtain amino-modified PCL powder. Step 3 Preparation of composite hydrogel: (1) Dissolve pepsin in 0.01 mol / mL dilute hydrochloric acid to prepare a 1 mg / mL pepsin hydrochloride solution, and filter with a 0.22 μm filter; add the sterilized DAT powder prepared in step 1 at 4°C and stir thoroughly for 48 hours until no powdery substance remains; (2) The solution prepared in (1) was adjusted to pH 7.4 with 0.1 mol / mL sodium hydroxide, and finally one-ninth of the total volume of 10× PBS was added and stored at 4°C; (3) The DAT hydrogel prepared in (2) was mixed with 1% of the PCL powder prepared in step 2 and cross-linked with 0.2% genipin to prepare a composite hydrogel.

8. The method for preparing the composite hydrogel according to claim 7, wherein: The sodium chloride solution in step 1 (6) contains 100 ug / mL DNase and 100 ug / mL RNase.

9. The method for preparing the composite hydrogel according to claim 7, wherein: In step 2 (1), the mass volume ratio of PCL to HFIP is 1:

10.

10. The method for preparing the composite hydrogel according to claim 7, characterized in that: The concentration of the DAT hydrogel in step three (3) is 8 mg / mL.

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