A porcine collagen chemical decellularization method based on supercritical fluid technology

Abstract

The application relates to the technical field of collagen, in particular to a pig collagen chemical decellularization method based on supercritical fluid technology, which comprises the following steps: taking fresh pig skin collagen, performing physical pretreatment after washing and cutting; placing the pretreated collagen in a supercritical reaction kettle, introducing supercritical fluid, controlling the pressure to be 30-42 MPa and the temperature to be 40-50 DEG C, maintaining the supercritical environment for 10-20 minutes; injecting a mixed reagent containing ethanol, protease and chelating agent into the supercritical reaction kettle, stirring for 1.0-2.0 hours; after the treatment is completed, the pressure of the reaction kettle is reduced in steps at a rate of 0.5-1 MPa / min, and supercritical fluid or mixed fluid of supercritical fluid and ethanol is introduced during the replacement, and finally the pressure is reduced to normal pressure; the collagen is taken out, subjected to enzymatic hydrolysis treatment, washed with a sterile solution, and the pig collagen is obtained. Through the supercritical multiphase fluid cooperation and nano-enzyme directional delivery technology, the DNA residual amount is greatly reduced.

Description

Technical Field

[0001] This invention relates to the field of collagen technology, specifically to a chemical decellularization method for porcine collagen based on supercritical fluid technology. Background Technology

[0002] Collagen, as a natural biomaterial, has broad application prospects in tissue engineering, regenerative medicine, and vascular embolization materials due to its excellent biocompatibility, biodegradability, and mechanical properties. Porcine collagen, in particular, is abundant and relatively inexpensive, making it a commonly used collagen raw material in clinical practice. However, incomplete removal of cellular components from natural porcine collagen can easily trigger immune rejection, severely limiting its clinical application. Furthermore, the structural stability, mechanical properties, and microenvironment compatibility of collagen directly affect its function; therefore, decellularization is necessary to optimize these properties.

[0003] Traditional chemical decellularization methods or single enzymatic hydrolysis methods often result in high levels of residual DNA when removing cell debris and DNA, which can easily trigger host immune rejection. Furthermore, the large amount of chemical reagents and enzymes remaining after treatment may lead to cytotoxicity and reduced biocompatibility. Prolonged enzymatic hydrolysis or high-temperature chemical treatment can easily damage the three-dimensional structure of collagen, leading to decreased mechanical properties such as tensile strength and elongation at break, reduced cross-linking degree, and difficulty in controlling the degradation rate. Simultaneously, collagen treated by existing methods has low porosity and a small average pore size, failing to provide an optimal microenvironment for cell adhesion and proliferation, thus affecting its application in tissue engineering. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by proposing a chemical decellularization method for porcine collagen based on supercritical fluid technology.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for chemical decellularization of porcine collagen based on supercritical fluid technology, comprising the following steps:

[0006] (1) Take fresh pig skin collagen, and after cleaning and cutting, perform physical pretreatment;

[0007] (2) Place the pretreated collagen in a supercritical reactor, introduce supercritical fluid, control the pressure at 30-42 MPa and the temperature at 40-50 °C, and maintain for 10-20 minutes to establish a supercritical environment;

[0008] (3) Inject a mixed reagent containing ethanol, protease and chelating agent into the supercritical reactor and stir for 1.0 to 2.0 hours;

[0009] (4) After the treatment is completed, the pressure of the reactor is reduced stepwise at a rate of 0.5 to 1 MPa / min. During this period, supercritical fluid or a mixture of supercritical fluid and ethanol is introduced for displacement, and finally reduced to atmospheric pressure.

[0010] (5) Remove the collagen, and after enzymatic hydrolysis, rinse with sterile solution.

[0011] Preferably, in step (1), the physical pretreatment is selected from at least one of the following:

[0012] Clean with physiological saline containing 0.1% heparin sodium using ultrasonic cleaning at a power of 250-350W and a frequency of 40-60kHz for 15-20 minutes, then freeze at -20℃ for 30 minutes;

[0013] Pre-treat with 0.02% Triton X-100 solution using ultrasound at 250W power and 50kHz frequency for 15 minutes;

[0014] The plasma was treated for 8 minutes in a low-temperature plasma treatment instrument with a power of 120W and an argon atmosphere at a distance of 5cm.

[0015] The sample was pretreated with a 0.01% graphene quantum dot solution by ultrasonication at 350W and 60kHz for 20 minutes, and then freeze-dried at -80℃ for 2 hours.

[0016] After soaking in an ethanol solution containing 0.05% fullerene for 15 minutes, it was pretreated with 300W / 40kHz ultrasound combined with 5MPa hydrostatic pressure for 10 minutes.

[0017] Preferably, in step (2), the supercritical fluid is supercritical CO2, or a mixture of supercritical CO2 and sulfur hexafluoride, wherein the volume ratio of CO2 to sulfur hexafluoride is 7:3.

[0018] Preferably, in step (3), the mixed reagent contains: ethanol with a mass fraction of 3% to 6.2%; the protease is selected from at least one of trypsin, bromelain, and papain, with a mass fraction of 0.18% to 0.25%; and the chelating agent is selected from at least one of EDTA, sodium citrate, potassium citrate, and sodium tartrate, with a mass fraction of 0.08% to 0.2%.

[0019] Preferably, in step (3), the stirring rate is 120-220 r / min; pulse pressure regulation can be applied during the process, specifically maintaining at 36 MPa for 20 minutes, then maintaining at 32 MPa for 10 minutes to complete one cycle, repeating 3 cycles; or maintaining at 38 MPa and 48°C for 15 minutes, then maintaining at 30 MPa and 42°C for 10 minutes to complete one cycle, repeating 4 cycles.

[0020] Preferably, in step (4), the stepped pressure reduction replacement includes:

[0021] First, reduce the pressure to 10 MPa at a rate of 0.5 MPa / min, then introduce pure supercritical CO2 for 30 minutes to replace the pressure.

[0022] Alternatively, the pressure can be reduced to 5 MPa at a rate of 1 MPa / min, and a supercritical CO2-ethanol mixture with a volume ratio of 9:1 can be introduced for 25 minutes to replace the pressure.

[0023] Alternatively, the pressure can be reduced to 20 MPa at a rate of 0.8 MPa / min, and a supercritical CO2-ethanol mixture with a volume ratio of 8:2 can be introduced for 40 minutes to replace the pressure. Then, the pressure can be reduced to 5 MPa at a rate of 0.5 MPa / min, and pure supercritical CO2 can be introduced for 30 minutes to replace the pressure.

[0024] Alternatively, the pressure can be reduced from 0.6 MPa / min to 15 MPa, and a supercritical CO2-sulfur hexafluoride mixed fluid with a volume ratio of 1:1 can be introduced for 25 minutes to replace the pressure. Then, the pressure can be reduced to 5 MPa, and supercritical CO2 can be introduced for 30 minutes to replace the pressure.

[0025] Preferably, in step (5), the enzymatic hydrolysis is performed by soaking in a PBS solution containing 50 U / mL DNase I for 30 minutes, or incubating in a PBS solution containing 100 U / mL nuclease at 37°C for 20 minutes, or ultrasonically washing in a PBS solution containing 0.05% dipotassium glycyrrhizate for 25 minutes.

[0026] Preferably, in step (3), the mixed reagent further includes an antioxidant, which is selected from at least one of tea polyphenols, vitamin C, and trehalose, with a mass fraction of 0.05%-0.12%.

[0027] Preferably, in step (3), the protease is chitosan-encapsulated trypsin with a nanoparticle size of 50-80 nm and a mass fraction of 0.2%.

[0028] Preferably, in step (3), 100W microwave assistance or dual-frequency ultrasonic assistance alternating between 20kHz / 500W and 80kHz / 300W can be applied during the processing, each lasting for 3 minutes.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] 1. This invention significantly reduces residual DNA levels through supercritical multiphase fluid synergy and nanozyme-directed delivery technology. In the examples, the residual DNA levels are significantly reduced compared to traditional chemical methods, effectively avoiding the risk of immune rejection.

[0031] 2. Employing a step-down pressure replacement and green reagent system, the residual ethanol content is significantly reduced; enzyme residues are virtually undetectable, solving the cytotoxicity problem of chemical reagents. Cell survival rates remain above 94%, reaching a maximum of 98.7%; collagen structure and performance are stable: through pulse pressure regulation and the synergistic effect of antioxidants, the tensile strength of collagen is increased to 9.1–11.0 MPa, and the elongation at break reaches 70.5%–82.1%, which are 10%–32% and 18%–38% higher than conventional enzymatic hydrolysis methods, respectively; the degree of cross-linking is maintained above 78.8%, and the degradation rate after 7 days is controlled at 11.8%–15.7%, significantly better than existing technologies, ensuring the structural stability of the material in vivo.

[0032] 2. Combining physical pretreatment with the permeability characteristics of supercritical fluids, the material porosity reaches 83.7%–90.7%, with an average pore size of 78.7–90.5 μm, providing an excellent microenvironment for cell adhesion and proliferation, improving efficiency by more than 70% compared to traditional methods; the processing time is shortened to 1.0–2.0 hours, only 12.5%–25% of that of conventional enzymatic hydrolysis; and a systematic range of process parameters is formed by adjusting the gradients of pressure, temperature, and reagent concentration, which can be flexibly controlled according to actual needs, making it suitable for industrial production.

[0033] Green and environmentally friendly: Supercritical fluids are used as solvents and extractants, with no volatile organic pollutant emissions throughout the process; natural antioxidants such as tea polyphenols and vitamin C are introduced to replace traditional chemical reagents, reducing environmental pollution and conforming to the concept of green manufacturing. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0035] Take fresh pig skin collagen, clean it with physiological saline containing 0.1% heparin sodium at 300W power and 40kHz frequency for 20 minutes, cut it into 1cm×1cm pieces, and freeze it at -20℃ for 30 minutes.

[0036] The pretreated collagen was placed in a supercritical reactor, and supercritical CO2 was introduced. The pressure was set to 32 MPa and the temperature to 40 °C, and the supercritical environment was established for 10 minutes.

[0037] The mixture of reagents (4.5% ethanol, 0.25% trypsin, and 0.1% EDTA by mass fraction) was injected using a high-pressure pump and continuously stirred at 150 rpm for 1.8 hours.

[0038] After the treatment is completed, the pressure in the reactor is first reduced to 10 MPa at a rate of 0.5 MPa / min, and then pure supercritical CO2 is introduced to replace it for 30 minutes, and then the pressure is naturally reduced to atmospheric pressure.

[0039] Remove the collagen, soak it in PBS solution containing 50 U / mL DNase I for 30 minutes, and finally rinse it 5 times with sterile physiological saline. Store at 4°C for later use. Example 2

[0040] Take fresh pig skin collagen, clean it with physiological saline containing 0.1% heparin sodium at 300W power and 40kHz frequency for 20 minutes, cut it into 1cm×1cm pieces, and freeze it at -20℃ for 30 minutes.

[0041] The pretreated collagen was placed in a supercritical reactor, and supercritical CO2 was introduced. The pressure was set to 30 MPa, which is 2 MPa lower than that in Example 1, and the temperature was set to 40°C. The supercritical environment was established by maintaining this setting for 10 minutes.

[0042] The mixture of reagents (4.5% ethanol, 0.25% trypsin, and 0.1% EDTA by mass fraction) was injected using a high-pressure pump and continuously stirred at 150 rpm for 1.8 hours.

[0043] After the treatment is completed, the pressure in the reactor is first reduced to 10 MPa at a rate of 0.5 MPa / min, and then pure supercritical CO2 is introduced to replace it for 30 minutes, and then the pressure is naturally reduced to atmospheric pressure.

[0044] Remove the collagen, soak it in PBS solution containing 50 U / mL DNase I for 30 minutes, and finally rinse it 5 times with sterile physiological saline. Store at 4°C for later use. Example 3

[0045] Take fresh pig skin collagen, clean it with physiological saline containing 0.1% heparin sodium at 300W power and 40kHz frequency for 20 minutes, cut it into 1cm×1cm pieces, and freeze it at -20℃ for 30 minutes.

[0046] The pretreated collagen was placed in a supercritical reactor, and supercritical CO2 was introduced. The pressure was set to 32 MPa and the temperature to 40 °C, and the supercritical environment was established for 10 minutes.

[0047] The mixed reagents were injected using a high-pressure pump: 5.0% ethanol, 0.25% trypsin, and 0.1% EDTA, with a mass fraction increased by 0.5% compared to Example 1. The mixture was then continuously stirred at 150 r / min for 1.8 hours.

[0048] After the treatment is completed, the pressure in the reactor is first reduced to 10 MPa at a rate of 0.5 MPa / min, and then pure supercritical CO2 is introduced to replace it for 30 minutes, and then the pressure is naturally reduced to atmospheric pressure.

[0049] Remove the collagen, soak it in PBS solution containing 50 U / mL DNase I for 30 minutes, and finally rinse it 5 times with sterile physiological saline. Store at 4°C for later use. Example 4

[0050] Fresh pigskin collagen was rinsed with physiological saline, then sonicated for 15 minutes with 0.02% Triton X-100 solution at 250W and 50kHz, and then cut into 1cm×1cm pieces.

[0051] Place the mixture into a supercritical reactor, introduce supercritical CO2, maintain a pressure of 36 MPa and a temperature of 42 °C, and stabilize for 20 minutes. Then, inject a mixed reagent: 6.2% ethanol, 0.18% trypsin, 0.1% sodium citrate, and 0.05% sodium tartrate.

[0052] Pulsed pressure control was used, maintaining 36 MPa for 20 minutes, followed by maintaining 32 MPa for 10 minutes, and the cycle was repeated 3 times, with a total processing time of 2.0 hours and a stirring rate of 120 r / min.

[0053] Pressure reduction process: The pressure is reduced to 5 MPa at a rate of 1 MPa / min, and a supercritical CO2-ethanol mixed fluid with a volume ratio of 9:1 is introduced for 25 minutes to replace the pressure, and then reduced to atmospheric pressure.

[0054] Collagen was neutralized with 0.1 mol / L glycine solution for 20 minutes and then rinsed four times with sterile saline to complete the treatment. Example 5

[0055] Fresh pigskin collagen was rinsed with physiological saline, then sonicated for 15 minutes with 0.02% Triton X-100 solution at 250W and 50kHz, and then cut into 1cm×1cm pieces.

[0056] Place it in a supercritical reactor, introduce supercritical CO2, maintain a pressure of 36 MPa and a temperature of 40°C (2°C lower than in Example 4), and stabilize for 20 minutes. Then, inject a mixed reagent: 6.2% ethanol, 0.18% trypsin, 0.1% sodium citrate, and 0.05% sodium tartrate.

[0057] Pulsed pressure control was used, maintaining 36 MPa for 20 minutes, followed by maintaining 32 MPa for 10 minutes, and the cycle was repeated 3 times, with a total processing time of 2.0 hours and a stirring rate of 120 r / min.

[0058] Pressure reduction process: The pressure is reduced to 5 MPa at a rate of 1 MPa / min, and a supercritical CO2-ethanol mixed fluid with a volume ratio of 9:1 is introduced for 25 minutes to replace the pressure, and then reduced to atmospheric pressure.

[0059] Collagen was neutralized with 0.1 mol / L glycine solution for 20 minutes and then rinsed four times with sterile saline to complete the treatment. Example 6

[0060] Fresh pigskin collagen was rinsed with physiological saline, then sonicated for 15 minutes with 0.02% Triton X-100 solution at 250W and 50kHz, and then cut into 1cm×1cm pieces.

[0061] The mixture was placed in a supercritical reactor and supercritical CO2 was introduced. After stabilizing at a pressure of 36 MPa and a temperature of 42 °C for 20 minutes, a mixed reagent was injected: 6.2% ethanol, 0.18% trypsin, 0.08% sodium citrate (0.02% lower than in Example 4), and 0.07% sodium tartrate (0.02% higher than in Example 4).

[0062] Pulsed pressure control was used, maintaining 36 MPa for 20 minutes, followed by 32 MPa for 10 minutes, and the cycle was repeated 3 times, with a total processing time of 2.0 hours and a stirring rate of 120 r / min.

[0063] Pressure reduction process: The pressure is reduced to 5 MPa at a rate of 1 MPa / min, and a supercritical CO2-ethanol mixed fluid with a volume ratio of 9:1 is introduced for 25 minutes to replace the pressure, and then reduced to atmospheric pressure.

[0064] Collagen was neutralized with 0.1 mol / L glycine solution for 20 minutes and then rinsed four times with sterile saline to complete the treatment. Example 7

[0065] Fresh pigskin collagen was rinsed with saline solution and then treated in a low-temperature plasma treatment instrument for 8 minutes at a power of 120W under an argon atmosphere at a distance of 5cm. It was then cut into 1cm x 1cm pieces.

[0066] The pretreated collagen was placed in a supercritical reactor, and a supercritical mixed fluid was introduced, with CO2:sulfur hexafluoride = 7:3. The pressure was set to 40 MPa and the temperature to 48 °C, and the supercritical environment was established for 15 minutes.

[0067] Inject the nanozyme mixture: 4% ethanol, 0.2% chitosan-encapsulated trypsin with a nanoparticle size of 50-80 nm, 0.12% hyaluronidase, and 0.08% trehalose. Start dynamic stirring at 200 r / min and apply 100W microwave assistance for 1.2 hours.

[0068] The pressure was reduced in a stepwise manner: the pressure was reduced to 20 MPa at a rate of 0.8 MPa / min, and a supercritical CO2-ethanol mixed fluid with a volume ratio of 8:2 was introduced for 40 minutes for displacement; then the pressure was reduced to 5 MPa at a rate of 0.5 MPa / min, and pure supercritical CO2 was introduced for 30 minutes for displacement, and finally the pressure was naturally reduced to atmospheric pressure.

[0069] Remove the collagen, sonicate it for 25 minutes with PBS solution containing 0.05% dipotassium glycyrrhizate, and store it aseptically at 4°C. Example 8

[0070] Fresh pigskin collagen was rinsed with saline solution and then treated in a low-temperature plasma treatment instrument for 8 minutes at a power of 120W under an argon atmosphere at a distance of 5cm. It was then cut into 1cm x 1cm pieces.

[0071] The pretreated collagen was placed in a supercritical reactor and a supercritical mixed fluid was introduced, with CO2:sulfur hexafluoride = 7:3. The pressure was set to 38 MPa, which is 2 MPa lower than that in Example 7, and the temperature was 48°C. The supercritical environment was established by maintaining this temperature for 15 minutes.

[0072] Inject the nanozyme mixture: 4% ethanol, 0.2% chitosan-encapsulated trypsin with a nanoparticle size of 50-80 nm, 0.12% hyaluronidase, and 0.08% trehalose. Start dynamic stirring at 200 r / min and apply 100W microwave assistance for 1.2 hours.

[0073] The pressure was reduced in a stepwise manner: the pressure was reduced to 20 MPa at a rate of 0.8 MPa / min, and a supercritical CO2-ethanol mixed fluid with a volume ratio of 8:2 was introduced for 40 minutes for displacement; then the pressure was reduced to 5 MPa at a rate of 0.5 MPa / min, and pure supercritical CO2 was introduced for 30 minutes for displacement, and finally the pressure was naturally reduced to atmospheric pressure.

[0074] Remove the collagen, sonicate it for 25 minutes with PBS solution containing 0.05% dipotassium glycyrrhizate, and store it aseptically at 4°C. Example 9

[0075] Fresh pigskin collagen was rinsed with saline solution and then treated in a low-temperature plasma treatment instrument for 8 minutes at a power of 120W under an argon atmosphere at a distance of 5cm. It was then cut into 1cm x 1cm pieces.

[0076] The pretreated collagen was placed in a supercritical reactor, and a supercritical mixed fluid was introduced, with CO2:sulfur hexafluoride = 7:3. The pressure was set to 40 MPa and the temperature to 48 °C, and the supercritical environment was established for 15 minutes.

[0077] Inject the nanozyme mixture: 3.5% ethanol (0.5% lower than in Example 7), 0.2% chitosan-encapsulated trypsin with a nanoparticle size of 50-80 nm, 0.12% hyaluronidase, and 0.08% trehalose. Start dynamic stirring at 200 r / min and apply 100 W microwave assistance for 1.2 hours.

[0078] The pressure was reduced in a stepwise manner: the pressure was reduced to 20 MPa at a rate of 0.8 MPa / min, and a supercritical CO2-ethanol mixed fluid with a volume ratio of 8:2 was introduced for 40 minutes for displacement; then the pressure was reduced to 5 MPa at a rate of 0.5 MPa / min, and pure supercritical CO2 was introduced for 30 minutes for displacement, and finally the pressure was naturally reduced to atmospheric pressure.

[0079] Remove the collagen, sonicate it for 25 minutes with PBS solution containing 0.05% dipotassium glycyrrhizate, and store it aseptically at 4°C. Example 10

[0080] Fresh pigskin collagen was pretreated with 0.01% graphene quantum dot solution by ultrasonication for 20 minutes at 350W and 60kHz, and then freeze-dried at -80℃ for 2 hours before being cut into 1cm×1cm pieces.

[0081] Placed in a supercritical reactor, a supercritical CO2 / ethanol mixed fluid with a volume ratio of 6:4 was introduced at a pressure of 38 MPa and a temperature of 48 °C. After stabilizing for 20 minutes, a composite reagent was injected: 4% glycerol, 0.22% bromelain, 0.15% EDTA-2Na, and 0.12% tea polyphenols.

[0082] Pulsed pressure-temperature coupling control was implemented, maintaining 38MPa / 48℃ for 15 minutes and 30MPa / 42℃ for 10 minutes, repeated 4 times, with a stirring rate of 180r / min and a total processing time of 1.5 hours.

[0083] Pressure reduction process: First, the temperature is reduced to 30℃, then the pressure is reduced to 10MPa at a rate of 1MPa / min, supercritical ethanol is introduced for replacement for 35 minutes, and finally the pressure is reduced to atmospheric pressure.

[0084] After soaking the collagen in a 0.02% chitosan solution for 30 minutes, rinse it 6 times with sterile deionized water and vacuum dry it for later use. Example 11

[0085] Fresh pigskin collagen was pretreated with 0.01% graphene quantum dot solution by ultrasonication for 20 minutes at 350W and 60kHz, and then freeze-dried at -80℃ for 2 hours before being cut into 1cm×1cm pieces.

[0086] The mixture was placed in a supercritical reactor and introduced with a supercritical CO2 / ethanol mixed fluid at a volume ratio of 6:4. The pressure was 38 MPa and the temperature was 50 °C, which was 2 °C higher than that in Example 10. After stabilizing for 20 minutes, a composite reagent was injected: 4% glycerol, 0.22% bromelain, 0.15% EDTA-2Na, and 0.12% tea polyphenols.

[0087] Pulsed pressure-temperature coupling control was implemented, maintaining 38 MPa and 50℃ for 15 minutes, followed by maintaining 30 MPa and 42℃ for 10 minutes, and the cycle was repeated 4 times. The stirring rate was 180 r / min, and the total treatment time was 1.5 hours.

[0088] Pressure reduction process: First, the temperature is reduced to 30℃, then the pressure is reduced to 10MPa at a rate of 1MPa / min, supercritical ethanol is introduced for replacement for 35 minutes, and finally the pressure is reduced to atmospheric pressure.

[0089] After soaking the collagen in a 0.02% chitosan solution for 30 minutes, rinse it 6 times with sterile deionized water and vacuum dry it for later use. Example 12

[0090] Fresh pigskin collagen was pretreated with 0.01% graphene quantum dot solution by ultrasonication for 20 minutes at 350W and 60kHz, and then freeze-dried at -80℃ for 2 hours before being cut into 1cm×1cm pieces.

[0091] The mixture was placed in a supercritical reactor and introduced with a supercritical CO2 / ethanol mixed fluid at a volume ratio of 6:4. The pressure was 38 MPa and the temperature was 48 °C. After stabilizing for 20 minutes, a composite reagent was injected: 4.5% glycerol (0.5% higher than in Example 10), 0.22% bromelain, 0.15% EDTA-2Na, and 0.12% tea polyphenols.

[0092] Pulsed pressure-temperature coupling control was implemented, maintaining 38 MPa and 48℃ for 15 minutes, followed by maintaining 30 MPa and 42℃ for 10 minutes, and the cycle was repeated 4 times. The stirring rate was 180 r / min, and the total treatment time was 1.5 hours.

[0093] Pressure reduction process: First, the temperature is reduced to 30℃, then the pressure is reduced to 10MPa at a rate of 1MPa / min, supercritical ethanol is introduced for replacement for 35 minutes, and finally the pressure is reduced to atmospheric pressure.

[0094] After soaking the collagen in a 0.02% chitosan solution for 30 minutes, rinse it 6 times with sterile deionized water and vacuum dry it for later use. Example 13

[0095] Fresh pigskin collagen was rinsed with saline solution, soaked in an ethanol solution containing 0.05% fullerene for 15 minutes, pretreated with ultrasound and high pressure for 10 minutes, and then cut into 1cm×1cm pieces using 300W / 40kHz ultrasound + 5MPa hydrostatic pressure.

[0096] Place the mixture into a supercritical reactor, introduce supercritical sulfur hexafluoride fluid, maintain a pressure of 42 MPa and a temperature of 50 °C for 12 minutes, and then inject the following reagents: 3% ethanol, 0.2% papain, 0.2% potassium citrate, and 0.05% vitamin C.

[0097] Dual-frequency ultrasonic assisted treatment was initiated, with alternating 20kHz / 500W and 80kHz / 300W for 3 minutes each, for a total treatment time of 1.0 hour and a stirring rate of 220r / min.

[0098] Pressure reduction and replacement: First, reduce the pressure to 15 MPa at 0.6 MPa / min, then introduce a supercritical CO2-sulfur hexafluoride mixed fluid at a volume ratio of 1:1 for 25 minutes; then reduce the pressure to 5 MPa, introduce supercritical CO2 for 30 minutes, and allow the pressure to decrease naturally.

[0099] After removal, incubate with PBS solution containing 100 U / mL nuclease at 37°C for 20 minutes, then rinse 5 times with sterile physiological saline to complete the treatment. Example 14

[0100] Fresh pigskin collagen was rinsed with saline solution, soaked in an ethanol solution containing 0.05% fullerene for 15 minutes, pretreated with ultrasound and high pressure for 10 minutes, and then cut into 1cm×1cm pieces using 300W / 40kHz ultrasound + 5MPa hydrostatic pressure.

[0101] Place the mixture into a supercritical reactor, introduce supercritical sulfur hexafluoride fluid, maintain a pressure of 40 MPa (2 MPa lower than in Example 13), and a temperature of 50°C for 12 minutes. Then inject the following reagents: 3% ethanol, 0.2% papain, 0.2% potassium citrate, and 0.05% vitamin C.

[0102] Dual-frequency ultrasonic assisted treatment was initiated, with alternating 20kHz / 500W and 80kHz / 300W for 3 minutes each, for a total treatment time of 1.0 hour and a stirring rate of 220r / min.

[0103] Pressure reduction and replacement: First, reduce the pressure to 15 MPa at 0.6 MPa / min, then introduce a supercritical CO2-sulfur hexafluoride mixed fluid at a volume ratio of 1:1 for 25 minutes; then reduce the pressure to 5 MPa, introduce supercritical CO2 for 30 minutes, and allow the pressure to decrease naturally.

[0104] After removal, incubate with PBS solution containing 100 U / mL nuclease at 37°C for 20 minutes, then rinse 5 times with sterile physiological saline to complete the treatment. Example 15

[0105] Fresh pigskin collagen was rinsed with saline solution, soaked in an ethanol solution containing 0.05% fullerene for 15 minutes, pretreated with ultrasound and high pressure for 10 minutes, and then cut into 1cm×1cm pieces using 300W / 40kHz ultrasound + 5MPa hydrostatic pressure.

[0106] Place the mixture into a supercritical reactor and introduce supercritical sulfur hexafluoride fluid. Maintain the pressure at 42 MPa and the temperature at 50 °C for 12 minutes, then inject the following reagents: 3% ethanol, 0.18% papain (0.02% lower than in Example 13), 0.2% potassium citrate, and 0.05% vitamin C.

[0107] Dual-frequency ultrasonic assisted treatment was initiated, with alternating 20kHz / 500W and 80kHz / 300W for 3 minutes each, for a total treatment time of 1.0 hour and a stirring rate of 220r / min.

[0108] Pressure reduction and replacement: First, reduce the pressure to 15 MPa at 0.6 MPa / min, then introduce a supercritical CO2-sulfur hexafluoride mixed fluid at a volume ratio of 1:1 for 25 minutes; then reduce the pressure to 5 MPa, introduce supercritical CO2 for 30 minutes, and allow the pressure to decrease naturally.

[0109] After removal, incubate with PBS solution containing 100 U / mL nuclease at 37°C for 20 minutes, then rinse 5 times with sterile physiological saline to complete the treatment.

[0110] Comparative Example 1

[0111] Take the same fresh pigskin collagen as in Example 1 and cut it into 1cm × 1cm pieces. Add 10% ethanol and 0.5% trypsin, and treat at 37°C for 4 hours based on collagen protein content. After treatment, rinse 3-5 times with physiological saline and set aside.

[0112] Comparative Example 2

[0113] Porcine collagen was treated with supercritical CO2 at 30 MPa / 40℃ for 2 hours without any added chemical reagents. After treatment, it was rinsed three times with physiological saline and then set aside.

[0114] Comparative Example 3

[0115] Small pieces of porcine collagen were treated with PBS solution containing 0.25% trypsin and 0.02% EDTA at 37°C for 8 hours by shaking. After rinsing five times with physiological saline, the mixture was ready for use.

[0116] Table 1 Test results of Examples 1-8

[0117]

[0118] Table 2 Test Results of Examples 9-15

[0119]

[0120] (1) The DNA residue levels in Examples 1 to 15 were significantly lower than those in Comparative Examples 1 to 3, with Example 7 showing the lowest value of 1.9 ng / mg, which was only 12.4% of that in Comparative Example 1. A regular pattern of change was observed within the same parameter gradient group: as seen in the comparison of Examples 1, 2, and 3, decreasing the pressure led to an increase in DNA residue, while increasing the ethanol concentration helped reduce residue, indicating that supercritical pressure and solvent concentration have a synergistic effect in enhancing decellularization. In Examples 13 to 15, after a pressure reduction of 2 MPa, the DNA residue level increased from 2.2 ng / mg to 2.6 ng / mg, while reducing the amount of papain also slightly increased the residue level, confirming the key influence of enzyme activity and fluid pressure on cell clearance.

[0121] (2) The cell survival rate of all examples remained above 93%, which was significantly higher than 75.6% of Comparative Example 1 and 82.3% of Comparative Example 2. In Examples 10 to 12, the cell survival rate increased from 98.1% to 98.3% after increasing the concentration of glycerol, indicating that the moisturizer can reduce collagen structural damage and thus improve biocompatibility. In Examples 7 to 9, although the DNA residue was slightly increased due to the decrease in ethanol concentration, the cell survival rate was still maintained above 97%, demonstrating the role of the nanozyme system in ensuring biocompatibility.

[0122] (3) The ethanol residue was lowest in Examples 13 to 15, with 2.4 μg / g in Example 15 being only 5.3% of that in Comparative Example 1. Examples 1 to 3 showed that increasing the ethanol concentration would increase the residue, but the supercritical displacement process could effectively alleviate this problem. Regarding enzyme residue, the trypsin residue in Examples 1 to 6 was all below 2 ng / g, which was 87.2% lower than the 15.6 ng / g in Comparative Example 3, indicating that the targeted removal ability of supercritical fluid is superior to that of traditional enzymatic hydrolysis systems.

[0123] (4) Regarding tensile strength, Example 13 showed the highest value of 11.0 MPa, which was 69.2% higher than Comparative Example 1, and the elongation at break was 1.65 times that of Comparative Example 1. The effect of parameter adjustment on mechanical properties showed a regularity: In Examples 4 to 6, adjusting the chelating agent ratio increased the tensile strength from 9.4 MPa to 9.5 MPa and the elongation at break from 71.5% to 72.0%, indicating that the composite chelating agent can reduce collagen fiber damage. In Examples 10 to 12, increasing the glycerol concentration increased the tensile strength from 10.5 MPa to 10.6 MPa, confirming the protective effect of the humectant on structural integrity.

[0124] (5) The 7-day degradation rate was the lowest in Examples 13 to 15, significantly lower than the 28.7% of Comparative Example 1, indicating that supercritical treatment can enhance the anti-degradation ability of collagen. In terms of porosity, Example 13 had the highest value of 90.7%, with an average pore size of 89.8 μm, providing an excellent microenvironment for cell adhesion, and its cell adhesion number was 26.8% higher than that of Comparative Example 3. In Examples 7 to 9, supercritical mixed fluid treatment maintained the porosity above 88.6%, which was better than Comparative Example 2 with single CO2 treatment.

[0125] (6) Through parameter correlation analysis, when the supercritical pressure is controlled at 38-42 MPa, the temperature at 42-48℃ and the ethanol concentration at 3.5%-5.0%, the comprehensive optimal performance of DNA residue ≤2.5 ng / mg, cell survival rate ≥97% and tensile strength ≥10 MPa can be achieved, corresponding to the core schemes of Examples 7, 10 and 13, which fully demonstrates the technical advantages of this invention in the synergistic optimization of multiple performance indicators.

[0126] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only to the claims and their full scope and equivalents.

Claims

1. A chemical decellularization method for porcine collagen based on supercritical fluid technology, characterized in that, The method includes the following steps: (1) Take fresh pig skin collagen, and after cleaning and cutting, perform physical pretreatment; (2) Place the pretreated collagen in a supercritical reactor, introduce supercritical fluid, control the pressure at 30-42 MPa and the temperature at 40-50 °C, and maintain for 10-20 minutes to establish a supercritical environment; (3) Inject a mixed reagent containing ethanol, protease and chelating agent into the supercritical reactor and stir for 1.0 to 2.0 hours; (4) After the treatment is completed, the pressure of the reactor is reduced stepwise at a rate of 0.5 to 1 MPa / min. During this period, supercritical fluid or a mixture of supercritical fluid and ethanol is introduced for replacement, and finally reduced to atmospheric pressure. (5) Remove the collagen, and after enzymatic hydrolysis, rinse with sterile solution.

2. The method according to claim 1, characterized in that, In step (1), the physical preprocessing is selected from at least one of the following: Clean with physiological saline containing 0.1% heparin sodium using ultrasonic cleaning at a power of 250-350W and a frequency of 40-60kHz for 15-20 minutes, then freeze at -20℃ for 30 minutes; Pre-treat with 0.02% Triton X-100 solution using ultrasound at 250W power and 50kHz frequency for 15 minutes; The plasma was treated for 8 minutes in a low-temperature plasma treatment instrument with a power of 120W and an argon atmosphere at a distance of 5cm. The sample was pretreated with a 0.01% graphene quantum dot solution by ultrasonication at 350W and 60kHz for 20 minutes, and then freeze-dried at -80℃ for 2 hours. After soaking in an ethanol solution containing 0.05% fullerene for 15 minutes, it was pretreated with 300W / 40kHz ultrasound combined with 5MPa hydrostatic pressure for 10 minutes.

3. The method according to claim 1, characterized in that, In step (2), the supercritical fluid is supercritical CO2, or a mixture of supercritical CO2 and sulfur hexafluoride, wherein the volume ratio of CO2 to sulfur hexafluoride is 7:

3.

4. The method according to claim 1, characterized in that, In step (3), the mass fraction of ethanol in the mixed reagent is 3% to 6.2%; The protease is selected from at least one of trypsin, bromelain, and papain, with a mass fraction of 0.18% to 0.25%. The chelating agent is selected from at least one of EDTA, sodium citrate, potassium citrate, and sodium tartrate, with a mass fraction of 0.08% to 0.2%.

5. The method according to claim 1, characterized in that, In step (3), the stirring rate is 120–220 r / min; During the treatment, pulsed pressure regulation was applied, specifically: maintaining at 36 MPa for 20 minutes, followed by maintaining at 32 MPa for 10 minutes to complete one cycle, and repeating the cycle 3 times; Alternatively, maintain at 38MPa and 48℃ for 15 minutes, then maintain at 30MPa and 42℃ for 10 minutes to complete one cycle, repeating 4 cycles.

6. The method according to claim 1, characterized in that, In step (4), the stepped pressure reduction replacement includes: First, reduce the pressure to 10 MPa at a rate of 0.5 MPa / min, then introduce pure supercritical CO2 for 30 minutes to replace the pressure. Alternatively, the pressure can be reduced to 5 MPa at a rate of 1 MPa / min, and a supercritical CO2-ethanol mixture with a volume ratio of 9:1 can be introduced for 25 minutes to replace the pressure. Alternatively, the pressure can be reduced to 20 MPa at a rate of 0.8 MPa / min, and a supercritical CO2-ethanol mixture with a volume ratio of 8:2 can be introduced for 40 minutes to replace the pressure. Then, the pressure can be reduced to 5 MPa at a rate of 0.5 MPa / min, and pure supercritical CO2 can be introduced for 30 minutes to replace the pressure. Alternatively, the pressure can be reduced from 0.6 MPa / min to 15 MPa, and a supercritical CO2-sulfur hexafluoride mixed fluid with a volume ratio of 1:1 can be introduced for 25 minutes to replace the pressure. Then, the pressure can be reduced to 5 MPa, and supercritical CO2 can be introduced for 30 minutes to replace the pressure.

7. The method according to claim 1, characterized in that, In step (5), the enzymatic hydrolysis is performed by soaking in PBS solution containing 50 U / mL DNase I for 30 minutes, or incubating in PBS solution containing 100 U / mL nuclease at 37°C for 20 minutes, or ultrasonically washing in PBS solution containing 0.05% dipotassium glycyrrhizate for 25 minutes.

8. The method according to claim 1, characterized in that, In step (3), the mixed reagent also includes an antioxidant, which is selected from at least one of tea polyphenols, vitamin C, and trehalose, with a mass fraction of 0.05%-0.12%.

9. The method according to claim 1, characterized in that, In step (3), the protease is chitosan-encapsulated trypsin with a nanoparticle size of 50-80 nm and a mass fraction of 0.2%.

10. The method according to claim 1, characterized in that, In step (3), 100W microwave assistance or dual-frequency ultrasound assistance alternating between 20kHz / 500W and 80kHz / 300W can also be applied during the treatment process, each lasting for 3 minutes.

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