CN113244463A - Method for manufacturing nano microcapsule acellular biological scaffold - Google Patents
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Abstract
The invention discloses a method for manufacturing a nano-microcapsule decellularized biological scaffold, which belongs to the technical field of biological engineering, and is characterized in that D-Hanks balanced salt buffer solution and TBS buffer solution are respectively used for treating bilateral carotid arteries of a white rabbit, pancreatin and complex enzyme are used for treating the bilateral carotid arteries to prepare a crude decellularized scaffold, and the crude decellularized scaffold is blended with a composite carrier to prepare the decellularized biological scaffold.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for manufacturing a nano microcapsule acellular biological scaffold.
Background
In tissue engineering, the selection of scaffold materials is one of the most important links, which is the basic structure for maintaining various functions in the place where seed cells live and after repair or reconstruction. An ideal vascular stent material should have the following properties: 1. the intravascular stent has good biocompatibility and no immunogenicity, can resist infection and can control the biodegradation speed, namely, the intravascular stent cannot form thrombus and the degradation speed is matched with the tissue growth; 2. the vascular stent has good affinity with cells and good mechanical properties, namely the surface of the vascular stent is suitable for cell attachment, proliferation and differentiation, can provide a suitable micro-stress environment for the cells, and has good compliance and durability; 3. the three-dimensional porous structure is suitable, namely the network structure inside the vascular stent needs to adapt to the growth of cells, the delivery of nutrients and the discharge of metabolites; 4. has proper callus capacity, can not induce inflammatory reaction and intimal hyperplasia, and can be well fused with surrounding tissues to form a part of self tissues; 5. finally, the material has easy processability and easy sterilization, namely the intravascular stent material is required to be suitable for production and manufacturing and cannot change the properties of the intravascular stent material due to ultraviolet irradiation or high temperature and high pressure during sterilization.
The existing nano microcapsule acellular biological scaffold has poor cell compatibility, and residual cell components after acellular process cannot be completely implanted into a body, so that calcification and tissue collapse are caused.
Disclosure of Invention
The invention aims to provide a method for manufacturing a nano microcapsule acellular biological scaffold.
The technical problems to be solved by the invention are as follows:
the existing nano microcapsule decellularized biological scaffold has poor cell compatibility, and residual cell components after decellularization can not be completely implanted into a body, so that calcification and tissue collapse are caused.
The purpose of the invention can be realized by the following technical scheme:
a method for manufacturing a nano microcapsule acellular biological scaffold specifically comprises the following steps:
step S1: taking down bilateral carotid arteries of white rabbits from big ears, trimming the adventitia, soaking in a D-Hanks balanced salt buffer solution for 10-15min at the temperature of 3-5 ℃, taking out the carotid arteries, soaking in a TBS buffer solution for 1-1.5h, taking out, adding into polyethylene glycol octyl phenyl ether, and carrying out ultrasonic treatment for 10-15h under the frequency of 3-5 MHz;
step S2: digesting carotid artery with pancreatin at 35-37 deg.C for 10-15h, adding complex enzyme, continuing to digest for 10-15h, washing with TBS buffer salt for 2-4 times, and freeze drying to obtain crude product of acellular scaffold;
step S3: dispersing the composite carrier in N, N-dimethylformamide to prepare carrier dispersion liquid, placing the acellular scaffold crude product in PBS buffer solution, adding the carrier dispersion liquid, uniformly dispersing, and drying at the temperature of 50-55 ℃ to prepare the acellular biological scaffold.
Further, the complex enzyme described in step S2 is prepared by mixing DNase and RNase in a mass ratio of 1: 1.
Further, the dosage mass ratio of the composite carrier and the crude acellular scaffold in the step S3 is 1: 5.
Further, the composite carrier is prepared by the following steps:
step A1: adding graphene into concentrated sulfuric acid, stirring for 6-10h under the conditions that the rotation speed is 200-10000 r/min and the temperature is 20-25 ℃, cooling to 10-15 ℃, adding potassium permanganate, stirring for 3-5min, stirring for 40-50min under the condition that the temperature is 70-80 ℃, adding deionized water, continuing stirring for 10-15min, heating to 100 ℃, refluxing and preserving heat for 10-20min, adding hydrogen peroxide, continuing stirring for 3-5min, centrifuging at the rotation speed of 8000-10000r/min to remove supernatant, and drying a substrate to obtain graphene oxide;
step A2: adding chitosan and dilute acetic acid into a reaction kettle, stirring for 10-15min under the condition that the rotation speed is 200-300r/min, adding a glyoxylic acid aqueous solution, reacting for 20-25h under the condition that the temperature is 25-30 ℃, adding sodium cyanoborohydride, continuing to react for 20-25h, filtering to remove a filtrate, rotating the filtrate in liquid nitrogen, and freeze-drying at the temperature of-80 ℃ to prepare carboxymethyl chitosan;
step A3: adding isophorone diisocyanate and ethyl acetate into a stirring kettle, stirring at the rotation speed of 150-200r/min and at the temperature of 70-80 ℃, adding polytetrahydrofuran diol and tetraphenyl tin, uniformly mixing, adding a dihydroxymethyl acrylic acid solution, reacting for 3-5 hours, cooling to 35-40 ℃, adding isopropanol, continuing to react for 20-30 minutes, adding ethylenediamine and 1-hydroxybenzotriazole, continuing to react for 1-1.5 hours, adding carboxymethyl chitosan, and reacting for 3-5 hours to obtain composite polyurethane;
step A4: soaking raw silk in diethyl ether for 3 times, soaking for 8h each time, drying after soaking, soaking the raw silk in ethanol for 20-25h, drying, adding into deionized water, boiling until sericin is completely removed to obtain sericin protein solution, adding composite polyurethane and graphene oxide into the sericin protein solution, performing ultrasonic treatment for 2-3h under the condition of frequency of 3-5MHz, and performing freeze-drying treatment to obtain the composite carrier.
Furthermore, the dosage ratio of the graphene, the concentrated sulfuric acid, the potassium permanganate, the deionized water and the hydrogen peroxide is 1g to 25mL to 3g to 150mL to 10mL, and the mass fraction of the concentrated sulfuric acid is 95%.
Further, the dosage ratio of the chitosan, the diluted acetic acid, the glyoxylic acid aqueous solution and the sodium cyanoborohydride is 1g to 100mL to 2mL to 0.15g, the mass fraction of the diluted acetic acid is 0.1-0.3%, and the mass fraction of the glyoxylic acid aqueous solution is 50%.
Furthermore, the using ratio of the isophorone diisocyanate, the ethyl acetate, the polytetrahydrofuran diol, the tetraphenyl tin, the dihydroxy methacrylic acid solution, the isopropanol, the ethylenediamine, the 1-hydroxybenzotriazole and the carboxymethyl chitosan is 2g to 10mL to 2.2g to 0.13 muL to 3mL to 0.75mL to 1.5mL to 0.35g to 0.8 g.
Furthermore, the dosage ratio of the raw silk to the deionized water is 1g:40mL, and the dosage ratio of the sericin solution, the composite polyurethane and the graphene oxide is 20mL:3g:5 g.
The invention has the beneficial effects that: the nano microcapsule decellularized biological scaffold prepared by the invention can reduce platelet adhesion, the strength of the decellularized scaffold can be increased and the self biocompatibility can be improved by taking the composite polyurethane as a scaffold part material, and the sericin has a good cell adhesion effect, so that the using effect of the nano microcapsule decellularized biological scaffold is better.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for manufacturing a nano microcapsule acellular biological scaffold specifically comprises the following steps:
step S1: taking down bilateral carotid arteries of a white rabbit with big ear, trimming the adventitia, soaking in a D-Hanks balanced salt buffer solution for 10min at the temperature of 3 ℃, taking out the carotid arteries, soaking in a TBS buffer solution for 1h, taking out the carotid arteries, adding into polyethylene glycol octyl phenyl ether, and carrying out ultrasonic treatment for 10h under the condition of the frequency of 3 MHz;
step S2: digesting carotid artery with pancreatin at 35 ℃ for 10h, adding complex enzyme, continuing to digest for 10h, washing for 2 times with TBS buffer salt, and freeze-drying after washing to obtain a crude product of the decellularized scaffold;
step S3: dispersing the composite carrier in N, N-dimethylformamide to prepare carrier dispersion liquid, placing the crude acellular scaffold in PBS buffer solution, adding the carrier dispersion liquid, uniformly dispersing, and drying at the temperature of 50 ℃ to prepare the acellular biological scaffold.
The composite carrier is prepared by the following steps:
step A1: adding graphene into concentrated sulfuric acid, stirring for 6 hours at the rotation speed of 200r/min and the temperature of 20 ℃, cooling to 10 ℃, adding potassium permanganate, stirring for 3 minutes, stirring for 40 minutes at the temperature of 70 ℃, adding deionized water, continuing stirring for 10 minutes, heating to 100 ℃, refluxing and preserving heat for 10 minutes, adding hydrogen peroxide, continuing stirring for 3 minutes, centrifuging at the rotation speed of 8000r/min to remove supernatant, and drying a substrate to obtain graphene oxide;
step A2: adding chitosan and dilute acetic acid into a reaction kettle, stirring for 10min at the rotation speed of 200r/min, adding a glyoxylic acid aqueous solution, reacting for 20h at the temperature of 25 ℃, adding sodium cyanoborohydride, continuing to react for 20h, filtering to remove a filtrate, rotating the filtrate in liquid nitrogen, and freeze-drying at the temperature of-80 ℃ to obtain carboxymethyl chitosan;
step A3: adding isophorone diisocyanate and ethyl acetate into a stirring kettle, stirring at the rotation speed of 150r/min and at the temperature of 70 ℃, adding polytetrahydrofuran diol and tetraphenyl tin, uniformly mixing, adding a dihydroxy methacrylic acid solution, reacting for 3 hours, cooling to the temperature of 35 ℃, adding isopropanol, continuing to react for 20 minutes, adding ethylenediamine and 1-hydroxybenzotriazole, continuing to react for 1 hour, adding carboxymethyl chitosan, and reacting for 3 hours to obtain composite polyurethane;
step A4: soaking raw silk in diethyl ether for 3 times, soaking for 8h each time, drying after soaking, soaking the raw silk in ethanol for 20h, drying, adding into deionized water, boiling until sericin is completely removed to obtain sericin protein solution, adding composite polyurethane and graphene oxide into the sericin protein solution, performing ultrasonic treatment for 2h under the condition of 3MHz, and performing freeze-drying treatment to obtain the composite carrier.
Example 2
A method for manufacturing a nano microcapsule acellular biological scaffold specifically comprises the following steps:
step S1: taking down bilateral carotid arteries of a white rabbit with big ear, trimming the adventitia, soaking in a D-Hanks balanced salt buffer solution for 10min at the temperature of 4 ℃, taking out the carotid arteries, soaking in a TBS buffer solution for 1h, taking out the carotid arteries, adding into polyethylene glycol octyl phenyl ether, and carrying out ultrasonic treatment for 10h under the condition of the frequency of 4 MHz;
step S2: digesting carotid artery with pancreatin at 36 ℃ for 10h, adding complex enzyme, continuing to digest for 10h, washing for 3 times with TBS buffer salt, and freeze-drying after washing to obtain a crude product of the decellularized scaffold;
step S3: dispersing the composite carrier in N, N-dimethylformamide to prepare carrier dispersion liquid, placing the crude acellular scaffold in PBS buffer solution, adding the carrier dispersion liquid, uniformly dispersing, and drying at the temperature of 50 ℃ to prepare the acellular biological scaffold.
The composite carrier is prepared by the following steps:
step A1: adding graphene into concentrated sulfuric acid, stirring for 6 hours at the rotation speed of 200r/min and the temperature of 23 ℃, cooling to the temperature of 13 ℃, adding potassium permanganate, stirring for 4 minutes, stirring for 40 minutes at the temperature of 75 ℃, adding deionized water, continuing stirring for 10 minutes, heating to the temperature of 100 ℃, refluxing and preserving heat for 10 minutes, adding hydrogen peroxide, continuing stirring for 3 minutes, centrifuging at the rotation speed of 8000r/min to remove supernatant, and drying a substrate to obtain graphene oxide;
step A2: adding chitosan and dilute acetic acid into a reaction kettle, stirring for 10min at the rotation speed of 200r/min, adding a glyoxylic acid aqueous solution, reacting for 20h at the temperature of 28 ℃, adding sodium cyanoborohydride, continuing to react for 20h, filtering to remove a filtrate, rotating the filtrate in liquid nitrogen, and freeze-drying at the temperature of-80 ℃ to obtain carboxymethyl chitosan;
step A3: adding isophorone diisocyanate and ethyl acetate into a stirring kettle, stirring at the rotation speed of 150r/min and at the temperature of 75 ℃, adding polytetrahydrofuran glycol and tetraphenyl tin, uniformly mixing, adding a dihydroxy methacrylic acid solution, reacting for 3 hours, cooling to the temperature of 38 ℃, adding isopropanol, continuing to react for 20 minutes, adding ethylenediamine and 1-hydroxybenzotriazole, continuing to react for 1 hour, adding carboxymethyl chitosan, and reacting for 3 hours to obtain composite polyurethane;
step A4: soaking raw silk in diethyl ether for 3 times, soaking for 8h each time, drying after soaking, soaking the raw silk in ethanol for 20h, drying, adding into deionized water, boiling until sericin is completely removed to obtain sericin protein solution, adding composite polyurethane and graphene oxide into the sericin protein solution, performing ultrasonic treatment for 2h under the condition of 4MHz, and performing freeze-drying treatment to obtain the composite carrier.
Example 3
A method for manufacturing a nano microcapsule acellular biological scaffold specifically comprises the following steps:
step S1: taking down bilateral carotid arteries of white rabbits from big ears, trimming the adventitia, soaking in a D-Hanks balanced salt buffer solution for 15min at the temperature of 4 ℃, taking out the carotid arteries, soaking in a TBS buffer solution for 1.5h, taking out, adding into polyethylene glycol octyl phenyl ether, and carrying out ultrasonic treatment for 15h under the condition of the frequency of 4 MHz;
step S2: digesting carotid artery with pancreatin at 36 ℃ for 15h, adding complex enzyme, continuing to digest for 15h, washing for 3 times with TBS buffer salt, and freeze-drying after washing to obtain a crude product of the decellularized scaffold;
step S3: dispersing the composite carrier in N, N-dimethylformamide to prepare carrier dispersion liquid, placing the crude acellular scaffold in PBS buffer solution, adding the carrier dispersion liquid, uniformly dispersing, and drying at the temperature of 55 ℃ to prepare the acellular biological scaffold.
The composite carrier is prepared by the following steps:
step A1: adding graphene into concentrated sulfuric acid, stirring for 10 hours at the rotation speed of 300r/min and the temperature of 23 ℃, cooling to the temperature of 13 ℃, adding potassium permanganate, stirring for 3-5 minutes, stirring for 50 minutes at the temperature of 75 ℃, adding deionized water, continuing stirring for 15 minutes, heating to the temperature of 100 ℃, refluxing and preserving heat for 20 minutes, adding hydrogen peroxide, continuing stirring for 5 minutes, centrifuging at the rotation speed of 10000r/min to remove supernatant, and drying a substrate to obtain graphene oxide;
step A2: adding chitosan and dilute acetic acid into a reaction kettle, stirring for 15min at the rotation speed of 300r/min, adding a glyoxylic acid aqueous solution, reacting for 25h at the temperature of 28 ℃, adding sodium cyanoborohydride, continuing to react for 25h, filtering to remove a filtrate, rotating the filtrate in liquid nitrogen, and freeze-drying at the temperature of-80 ℃ to obtain carboxymethyl chitosan;
step A3: adding isophorone diisocyanate and ethyl acetate into a stirring kettle, stirring at the rotation speed of 200r/min and at the temperature of 75 ℃, adding polytetrahydrofuran diol and tetraphenyl tin, uniformly mixing, adding a dihydroxy methacrylic acid solution, reacting for 5 hours, cooling to the temperature of 38 ℃, adding isopropanol, continuing to react for 30 minutes, adding ethylenediamine and 1-hydroxybenzotriazole, continuing to react for 1.5 hours, adding carboxymethyl chitosan, and reacting for 3-5 hours to obtain composite polyurethane;
step A4: soaking raw silk in diethyl ether for 3 times, soaking for 8h each time, drying after soaking, soaking the raw silk in ethanol for 25h, drying, adding into deionized water, boiling until sericin is completely removed to obtain sericin protein solution, adding composite polyurethane and graphene oxide into the sericin protein solution, performing ultrasonic treatment for 3h under the condition of 4MHz, and performing freeze-drying treatment to obtain the composite carrier.
Example 4
A method for manufacturing a nano microcapsule acellular biological scaffold specifically comprises the following steps:
step S1: taking down bilateral carotid arteries of white rabbits from big ears, trimming the adventitia, soaking in a D-Hanks balanced salt buffer solution for 15min at the temperature of 5 ℃, taking out the carotid arteries, soaking in a TBS buffer solution for 1.5h, taking out, adding into polyethylene glycol octyl phenyl ether, and carrying out ultrasonic treatment for 15h under the condition of the frequency of 5 MHz;
step S2: digesting carotid artery with pancreatin at 37 ℃ for 15h, adding complex enzyme, continuing to digest for 15h, washing with TBS buffer salt for 4 times, and freeze-drying after washing to obtain a crude product of the decellularized scaffold;
step S3: dispersing the composite carrier in N, N-dimethylformamide to prepare carrier dispersion liquid, placing the crude acellular scaffold in PBS buffer solution, adding the carrier dispersion liquid, uniformly dispersing, and drying at the temperature of 55 ℃ to prepare the acellular biological scaffold.
The composite carrier is prepared by the following steps:
step A1: adding graphene into concentrated sulfuric acid, stirring for 10 hours at the rotation speed of 300r/min and the temperature of 25 ℃, cooling to 15 ℃, adding potassium permanganate, stirring for 5 minutes, stirring for 50 minutes at the temperature of 80 ℃, adding deionized water, continuing stirring for 15 minutes, heating to 100 ℃, refluxing and preserving heat for 20 minutes, adding hydrogen peroxide, continuing stirring for 5 minutes, centrifuging at the rotation speed of 10000r/min to remove supernatant, and drying a substrate to obtain graphene oxide;
step A2: adding chitosan and dilute acetic acid into a reaction kettle, stirring for 15min at the rotation speed of 300r/min, adding a glyoxylic acid aqueous solution, reacting for 25h at the temperature of 30 ℃, adding sodium cyanoborohydride, continuing to react for 25h, filtering to remove a filtrate, rotating the filtrate in liquid nitrogen, and freeze-drying at the temperature of-80 ℃ to obtain carboxymethyl chitosan;
step A3: adding isophorone diisocyanate and ethyl acetate into a stirring kettle, stirring at the rotation speed of 200r/min and at the temperature of 80 ℃, adding polytetrahydrofuran diol and tetraphenyl tin, uniformly mixing, adding a dihydroxy methacrylic acid solution, reacting for 5 hours, cooling to the temperature of 40 ℃, adding isopropanol, continuing to react for 30 minutes, adding ethylenediamine and 1-hydroxybenzotriazole, continuing to react for 1.5 hours, adding carboxymethyl chitosan, and reacting for 5 hours to obtain composite polyurethane;
step A4: soaking raw silk in diethyl ether for 3 times, soaking for 8h each time, drying after soaking, soaking the raw silk in ethanol for 25h, drying, adding into deionized water, boiling until sericin is completely removed to obtain sericin protein solution, adding composite polyurethane and graphene oxide into the sericin protein solution, performing ultrasonic treatment for 3h under the condition of 5MHz, and performing freeze-drying treatment to obtain the composite carrier.
The foregoing is merely exemplary and illustrative of the principles of the present invention and various modifications, additions and substitutions of the specific embodiments described herein may be made by those skilled in the art without departing from the principles of the present invention or exceeding the scope of the claims set forth herein.
Claims (8)
1. A method for manufacturing a nano microcapsule acellular biological scaffold is characterized by comprising the following steps: the method specifically comprises the following steps:
step S1: taking down bilateral carotid arteries of white rabbits from big ears, trimming the adventitia, soaking in a D-Hanks balanced salt buffer solution for 10-15min at the temperature of 3-5 ℃, taking out the carotid arteries, soaking in a TBS buffer solution for 1-1.5h, taking out, adding into polyethylene glycol octyl phenyl ether, and carrying out ultrasonic treatment for 10-15h under the frequency of 3-5 MHz;
step S2: digesting carotid artery with pancreatin at 35-37 deg.C for 10-15h, adding complex enzyme, continuing to digest for 10-15h, washing with TBS buffer salt for 2-4 times, and freeze drying to obtain crude product of acellular scaffold;
step S3: dispersing the composite carrier in N, N-dimethylformamide to prepare carrier dispersion liquid, placing the acellular scaffold crude product in PBS buffer solution, adding the carrier dispersion liquid, uniformly dispersing, and drying at the temperature of 50-55 ℃ to prepare the acellular biological scaffold.
2. The method for preparing a nanocapsule acellular biological scaffold according to claim 1, wherein the nanocapsule acellular biological scaffold comprises: the complex enzyme described in the step S2 is formed by mixing DNase and RNase in a mass ratio of 1: 1.
3. The method for preparing a nanocapsule acellular biological scaffold according to claim 1, wherein the nanocapsule acellular biological scaffold comprises: the dosage mass ratio of the composite carrier to the crude cell-free scaffold product in the step S3 is 1: 5.
4. The method for preparing a nanocapsule acellular biological scaffold according to claim 1, wherein the nanocapsule acellular biological scaffold comprises: the composite carrier is prepared by the following steps:
step A1: adding graphene into concentrated sulfuric acid, stirring for 6-10h under the conditions that the rotation speed is 200-10000 r/min and the temperature is 20-25 ℃, cooling to 10-15 ℃, adding potassium permanganate, stirring for 3-5min, stirring for 40-50min under the condition that the temperature is 70-80 ℃, adding deionized water, continuing stirring for 10-15min, heating to 100 ℃, refluxing and preserving heat for 10-20min, adding hydrogen peroxide, continuing stirring for 3-5min, centrifuging at the rotation speed of 8000-10000r/min to remove supernatant, and drying a substrate to obtain graphene oxide;
step A2: adding chitosan and dilute acetic acid into a reaction kettle, stirring for 10-15min under the condition that the rotation speed is 200-300r/min, adding a glyoxylic acid aqueous solution, reacting for 20-25h under the condition that the temperature is 25-30 ℃, adding sodium cyanoborohydride, continuing to react for 20-25h, filtering to remove a filtrate, rotating the filtrate in liquid nitrogen, and freeze-drying at the temperature of-80 ℃ to prepare carboxymethyl chitosan;
step A3: adding isophorone diisocyanate and ethyl acetate into a stirring kettle, stirring at the rotation speed of 150-200r/min and at the temperature of 70-80 ℃, adding polytetrahydrofuran diol and tetraphenyl tin, uniformly mixing, adding a dihydroxymethyl acrylic acid solution, reacting for 3-5 hours, cooling to 35-40 ℃, adding isopropanol, continuing to react for 20-30 minutes, adding ethylenediamine and 1-hydroxybenzotriazole, continuing to react for 1-1.5 hours, adding carboxymethyl chitosan, and reacting for 3-5 hours to obtain composite polyurethane;
step A4: soaking raw silk in diethyl ether for 3 times, soaking for 8h each time, drying after soaking, soaking the raw silk in ethanol for 20-25h, drying, adding into deionized water, boiling until sericin is completely removed to obtain sericin protein solution, adding composite polyurethane and graphene oxide into the sericin protein solution, performing ultrasonic treatment for 2-3h under the condition of frequency of 3-5MHz, and performing freeze-drying treatment to obtain the composite carrier.
5. The method for preparing a nanocapsule acellular biological scaffold according to claim 4, wherein the nanocapsule acellular biological scaffold comprises: the dosage ratio of the graphene to the concentrated sulfuric acid to the potassium permanganate to the deionized water to the hydrogen peroxide is 1g to 25mL to 3g to 150mL to 10mL, and the mass fraction of the concentrated sulfuric acid is 95%.
6. The method for preparing a nanocapsule acellular biological scaffold according to claim 4, wherein the nanocapsule acellular biological scaffold comprises: the dosage ratio of the chitosan, the diluted acetic acid, the glyoxylic acid aqueous solution and the sodium cyanoborohydride is 1g to 100mL to 2mL to 0.15g, the mass fraction of the diluted acetic acid is 0.1-0.3%, and the mass fraction of the glyoxylic acid aqueous solution is 50%.
7. The method for preparing a nanocapsule acellular biological scaffold according to claim 4, wherein the nanocapsule acellular biological scaffold comprises: the using amount ratio of the isophorone diisocyanate, the ethyl acetate, the polytetrahydrofuran diol, the tetraphenyl tin, the dihydroxy methacrylic acid solution, the isopropanol, the ethylenediamine, the 1-hydroxybenzotriazole and the carboxymethyl chitosan is 2g to 10mL to 2.2g to 0.13 muL to 3mL to 0.75mL to 1.5mL to 0.35g to 0.8 g.
8. The method for preparing a nanocapsule acellular biological scaffold according to claim 4, wherein the nanocapsule acellular biological scaffold comprises: the dosage ratio of the raw silk to the deionized water is 1g:40mL, and the dosage ratio of the sericin solution, the composite polyurethane and the graphene oxide is 20mL:3g:5 g.
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