Disclosure of Invention
The invention aims to provide a decellularized matrix material and a preparation method and application thereof, and the preparation method has the advantages of low cost, wide raw material sources and simple preparation method, solves the problems of high degradation rate, incapacity of regulation and control, poor mechanical property, easiness in initiating inflammatory reaction and the like of the traditional decellularized matrix material, has excellent biocompatibility, low immunogenicity and bioactivity, reduces the degradation rate of products and improves the mechanical strength of the products.
The technical scheme of the invention is realized as follows:
the invention provides a preparation method of a decellularized matrix material, which comprises the steps of performing pretreatment on pigskin, performing decellularization treatment in an ionic liquid filled with carbon dioxide, performing enzymolysis to remove surface antigens, and reacting with ferulic acid and hyaluronic acid/antibacterial peptide complex to obtain the decellularized matrix material.
As a further improvement of the invention, the method comprises the following steps:
S1, pretreatment of pigskin: cleaning pigskin, dehairing, cleaning, removing epidermis cuticle and subcutaneous fat layer, taking dermis layer, cutting into proper size, soaking with 2-4% sodium hydroxide, and oscillating for 3-10h to obtain pretreated pigskin material;
S2, cell removal treatment: adding the pretreated pigskin material into the ionic liquid, introducing carbon dioxide, stirring, dialyzing, and freeze-drying to obtain a acellular dermal matrix;
S3, enzymolysis treatment: adding the acellular dermal matrix into water, adding alpha-galactosidase and trypsin, heating for enzymolysis, filtering, washing, and drying to obtain the acellular dermal matrix;
S4, preparation of hyaluronic acid/antibacterial peptide complex: adding hyaluronic acid into water, adding EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide) and NHS (N-hydroxysuccinimide), performing an activation reaction, adding antibacterial peptide, stirring, performing a reaction, dialyzing, and freeze-drying to obtain hyaluronic acid/antibacterial peptide complex;
S5, preparing a decellularized matrix material: mixing ferulic acid and acellular dermal matrix, adding EDC and NHS, activating, adding hyaluronic acid/antibacterial peptide complex, stirring, dialyzing, and lyophilizing.
As a further improvement of the invention, the solid-to-liquid ratio of the pretreated pigskin material and the ionic liquid in the step S2 is 1:7-10g/mL, the ventilation amount of the carbon dioxide is 20-30mL/min, the stirring treatment time is 1-2h, and the ionic liquid is at least one selected from acetic acid-3-methylimidazole hexafluorophosphate, 1-propyl-3-methylimidazole hexafluorophosphate, boron pentafluoroanthraquinone ketone ionic liquid, N-butylpyridine tetrafluoroborate, 4-formamidinopyridine hydrochloride, pyridine p-toluenesulfonate, 1-butyl-2, 3-dimethylimidazole hexafluorophosphate and 1-butyl-2, 3-dimethylimidazole trifluoromethane sulfonate.
As a further improvement of the invention, the ionic liquid is a mixture of 1-propyl-3-methylimidazole hexafluorophosphate and 1-butyl-2, 3-dimethylimidazole trifluoromethane sulfonate, and the mass ratio is 10:3-5.
As a further improvement of the invention, the mass ratio of the acellular dermal matrix, the alpha-galactosidase and the trypsin in the step S3 is 100:3-5:2-3, and the temperature of the heating enzymolysis is 35-38 ℃ for 2-4h.
As a further improvement of the invention, the mass ratio of the hyaluronic acid, EDC, NHS and the antibacterial peptide in the step S4 is 10:4-6:2-3:5-7, the temperature of the activation reaction is 20-25 ℃, the time is 30-60min, and the stirring reaction time is 12-15h.
As a further improvement of the invention, in the step S5, the mass ratio of the ferulic acid, the antigen-removing decellularized dermal matrix, the EDC, the NHS and the hyaluronic acid/antibacterial peptide complex is 2-4:12-15:6-8:3-4:7-9, the temperature of the activation reaction is 0-4 ℃ for 30-50min, and the stirring reaction time is 20-24h.
As a further improvement of the invention, the method specifically comprises the following steps:
S1, pretreatment of pigskin: cleaning pigskin, dehairing, cleaning, removing epidermis cuticle and subcutaneous fat layer, taking dermis layer, cutting into proper size, soaking with 2-4% sodium hydroxide, and oscillating for 3-10h to obtain pretreated pigskin material;
S2, cell removal treatment: adding the pretreated pigskin material into ionic liquid, wherein the solid-to-liquid ratio of the pretreated pigskin material to the ionic liquid is 1:7-10g/mL, introducing carbon dioxide, introducing ventilation amount of 20-30mL/min, stirring for 1-2h, dialyzing, and freeze-drying to obtain the acellular dermal matrix;
S3, enzymolysis treatment: adding 100 parts by weight of acellular dermal matrix into water, adding 3-5 parts by weight of alpha-galactosidase and 2-3 parts by weight of trypsin, heating to 35-38 ℃, performing enzymolysis for 2-4 hours, filtering, washing, and drying to obtain the acellular dermal matrix;
S4, preparation of hyaluronic acid/antibacterial peptide complex: adding 10 parts by weight of hyaluronic acid into water, adding 4-6 parts by weight of EDC and 2-3 parts by weight of NHS, performing activation reaction at 20-25 ℃ for 30-60min, adding 5-7 parts by weight of antibacterial peptide, stirring and reacting for 12-15h, dialyzing, and freeze-drying to obtain hyaluronic acid/antibacterial peptide compound;
S5, preparing a decellularized matrix material: mixing 2-4 parts by weight of ferulic acid and 12-15 parts by weight of antigen-removing acellular dermal matrix, adding 6-8 parts by weight of EDC and 3-4 parts by weight of NHS, performing an activation reaction at 0-4 ℃ for 30-50min, adding 7-9 parts by weight of hyaluronic acid/antibacterial peptide complex, stirring and reacting for 20-24h, dialyzing, and freeze-drying to obtain the acellular matrix material.
The invention further provides the acellular matrix material prepared by the preparation method.
The invention further protects application of the acellular matrix material in preparing medical products.
The invention has the following beneficial effects:
The invention prepares the acellular matrix material, and solves the problems of high degradation rate, incapacity of regulation and control, poor mechanical property, easiness in triggering inflammatory reaction and the like of the traditional acellular matrix material.
The invention firstly pretreats pigskin to obtain pigskin material with dermis layer, extracts the pigskin material in the ionic liquid which is introduced with carbon dioxide, removes cells, removal of cells by conventional chemical, biological, etc. methods may cause some key substances, such as glycoproteins and growth factors, to be denatured or removed by the surface-active action of the detergent. The method for removing the cells has excellent biocompatibility, low immunogenicity and bioactivity, CO2 is usually nonpolar, can dissolve polar substances which are usually indissolvable in nonpolar solvents after being introduced into ionic liquid, permeates into tissues through CO2, and dissolves partial components in the cells by utilizing the dissolving capacity of the polar substances, so that the removal of the cell contents is realized, in addition, CO2 can increase the fluidity of the cell membranes, accelerate the rupture of the cell membranes, release the cell contents and further promote the cell removal process. The use of chemical reagents is not involved, so that DNA can be removed, the skeleton structure of the DNA can be reserved to a large extent, and meanwhile, the participation of ionic liquid can assist in removing all cell components in tissues, so that the decellularization efficiency is greatly improved.
However, the acellular dermal matrix obtained by the method has a small amount of heterogeneous antigen (such as alpha-Gal antigen) which can cause stronger heterogeneous immune response, the invention adopts alpha-galactosidase to remove alpha-Gal antigen, and cooperates with trypsin to remove other protein antigen substances, thereby greatly reducing inflammatory reaction caused by the prepared product.
In addition, the hyaluronic acid and the antibacterial peptide are compounded to prepare the compound, which has the functions of resisting infection and regenerating and repairing, and the hyaluronic acid and the antibacterial peptide are fixed on the acellular dermal matrix together, so that the anti-inflammatory, antibacterial and anti-infection effects of the material are greatly improved, the occurrence of immune reaction is reduced, the red swelling phenomenon is reduced, and the reactions such as allergy and the like are not easy to occur.
However, the prepared material still has the problems of high degradation rate, uncontrollable degradation rate and poor mechanical property, and the inventor surprisingly found that in the process of using NHS and EDC for activation, the addition of EDC and NHS can also induce intramolecular/intermolecular crosslinking of collagen in the catalytic decellularized matrix material, so that the enzymolysis resistance of the material is improved, the crosslinking property of the material is improved, the activator not only has better biocompatibility, but also can regulate and control the degradation rate of the material, solve the problems of high degradation rate and uncontrollable degradation rate of the non-crosslinked material, and improve the mechanical strength of the product.
In addition, the acellular matrix material prepared by the invention can be degraded to generate glycyl-histidyl-lysine tripeptide, and has the effects of promoting synthesis of collagen, mucopolysaccharide and proteoglycan, stimulating the activity of prolyl enzyme, promoting biological functions such as angiogenesis and accelerating wound healing.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Alpha-galactosidase, 1 ten thousand U/g, purchased from merck life sciences; trypsin, 5000U/g, purchased from merck life sciences; hyaluronic acid, >99%, purchased from west An Purui s bioengineering, inc; antibacterial peptides were purchased from hubei wedeli chemical technologies limited.
1-Propyl-3-methylimidazole hexafluorophosphate and 1-butyl-2, 3-dimethylimidazole trifluoromethane sulfonate, purchased from microphone agents.
Example 1
The embodiment provides a preparation method of a decellularized matrix material, which specifically comprises the following steps:
S1, pretreatment of pigskin: cleaning pigskin, dehairing, cleaning, removing epidermis cuticle and subcutaneous fat layer, taking dermis layer, cutting into proper size, soaking with 2% sodium hydroxide solution, and oscillating for 10h to obtain pretreated pigskin material;
S2, cell removal treatment: adding the pretreated pigskin material into ionic liquid, wherein the solid-to-liquid ratio of the pretreated pigskin material to the ionic liquid is 1:7g/mL, introducing carbon dioxide, and the ventilation amount is 20mL/min, stirring for 2h, dialyzing, and freeze-drying to obtain a acellular dermal matrix;
the ionic liquid is a mixture of 1-propyl-3-methylimidazole hexafluorophosphate and 1-butyl-2, 3-dimethylimidazole trifluoromethane sulfonate, and the mass ratio is 10:3;
S3, enzymolysis treatment: adding 100g of acellular dermal matrix into 500mL of water, adding 3g of alpha-galactosidase and 2g of trypsin, heating to 35 ℃, carrying out enzymolysis for 4 hours, filtering, washing and drying to obtain the acellular dermal matrix;
S4, preparation of hyaluronic acid/antibacterial peptide complex: adding 10g of hyaluronic acid into 200mL of water, adding 4gEDC g of NHS and 2g of NHS, performing an activation reaction at 20 ℃ for 30min, adding 5g of antibacterial peptide, stirring and reacting for 12h, dialyzing, and freeze-drying to obtain hyaluronic acid/antibacterial peptide compound;
S5, preparing a decellularized matrix material: mixing 2g of ferulic acid and 12g of antigen-free acellular dermal matrix, adding 200mL of water, adding 6g of EDC and 3g of NHS, performing activation reaction at 0 ℃ for 30min, adding 7g of hyaluronic acid/antibacterial peptide complex, stirring, reacting for 20h, dialyzing, and freeze-drying to obtain the acellular matrix material.
Example 2
The embodiment provides a preparation method of a decellularized matrix material, which specifically comprises the following steps:
s1, pretreatment of pigskin: cleaning pigskin, dehairing, cleaning, removing epidermis cuticle and subcutaneous fat layer, taking dermis layer, cutting into proper size, soaking with 4% sodium hydroxide solution, and oscillating for 3h to obtain pretreated pigskin material;
S2, cell removal treatment: adding the pretreated pigskin material into ionic liquid, wherein the solid-to-liquid ratio of the pretreated pigskin material to the ionic liquid is 1:10g/mL, introducing carbon dioxide, and the ventilation amount is 30mL/min, stirring for 1h, dialyzing, and freeze-drying to obtain a acellular dermal matrix;
The ionic liquid is a mixture of 1-propyl-3-methylimidazole hexafluorophosphate and 1-butyl-2, 3-dimethylimidazole trifluoromethane sulfonate, and the mass ratio is 10:5;
S3, enzymolysis treatment: adding 100g of acellular dermal matrix into 500mL of water, adding 5g of alpha-galactosidase and 3g of trypsin, heating to 38 ℃, carrying out enzymolysis for 2 hours, filtering, washing and drying to obtain the acellular dermal matrix;
S4, preparation of hyaluronic acid/antibacterial peptide complex: adding 10g of hyaluronic acid into 200mL of water, adding 6gEDC and 3g of NHS, performing activation reaction at 25 ℃ for 60min, adding 7g of antibacterial peptide, stirring for reaction for 15h, dialyzing, and freeze-drying to obtain hyaluronic acid/antibacterial peptide compound;
s5, preparing a decellularized matrix material: mixing 4g of ferulic acid and 15g of antigen-free acellular dermal matrix, adding into 200mL of water, adding 8g of EDC and 4g of NHS, performing activation reaction at 4 ℃ for 50min, adding 9g of hyaluronic acid/antibacterial peptide complex, stirring, reacting for 24h, dialyzing, and freeze-drying to obtain the acellular matrix material.
Example 3
The embodiment provides a preparation method of a decellularized matrix material, which specifically comprises the following steps:
s1, pretreatment of pigskin: cleaning pigskin, dehairing, cleaning, removing epidermis cuticle and subcutaneous fat layer, taking dermis layer, cutting into proper size, soaking with 3% sodium hydroxide solution, and oscillating for 5h to obtain pretreated pigskin material;
s2, cell removal treatment: adding the pretreated pigskin material into ionic liquid, wherein the solid-to-liquid ratio of the pretreated pigskin material to the ionic liquid is 1:8g/mL, introducing carbon dioxide, and the ventilation amount is 25mL/min, stirring for 1.5h, dialyzing, and freeze-drying to obtain a acellular dermal matrix;
The ionic liquid is a mixture of 1-propyl-3-methylimidazole hexafluorophosphate and 1-butyl-2, 3-dimethylimidazole trifluoromethane sulfonate, and the mass ratio is 10:4;
S3, enzymolysis treatment: adding 100g of acellular dermal matrix into 500mL of water, adding 4g of alpha-galactosidase and 2.5g of trypsin, heating to 37 ℃, carrying out enzymolysis for 3 hours, filtering, washing and drying to obtain the acellular dermal matrix;
s4, preparation of hyaluronic acid/antibacterial peptide complex: adding 10g of hyaluronic acid into 200mL of water, adding 5gEDC g of NHS and 2.5g of NHS, performing activation reaction at 22 ℃ for 45min, adding 6g of antibacterial peptide, stirring and reacting for 13h, dialyzing, and freeze-drying to obtain hyaluronic acid/antibacterial peptide compound;
S5, preparing a decellularized matrix material: 3g of ferulic acid and 13g of antigen-free acellular dermal matrix are mixed and added into 200mL of water, 7g of EDC and 3.5g of NHS are added, activation reaction is carried out for 40min at 2 ℃, 8g of hyaluronic acid/antibacterial peptide complex is added, stirring reaction is carried out for 22h, dialysis and freeze drying are carried out, and the acellular matrix material is prepared.
Comparative example 1
In contrast to example 3, carbon dioxide was not introduced in step S2.
The method comprises the following steps:
s1, pretreatment of pigskin: cleaning pigskin, dehairing, cleaning, removing epidermis cuticle and subcutaneous fat layer, taking dermis layer, cutting into proper size, soaking with 3% sodium hydroxide solution, and oscillating for 5h to obtain pretreated pigskin material;
s2, cell removal treatment: adding the pretreated pigskin material into ionic liquid, wherein the solid-to-liquid ratio of the pretreated pigskin material to the ionic liquid is 1:8g/mL, stirring for 1.5h, dialyzing, and freeze-drying to obtain a acellular dermal matrix;
The ionic liquid is a mixture of 1-propyl-3-methylimidazole hexafluorophosphate and 1-butyl-2, 3-dimethylimidazole trifluoromethane sulfonate, and the mass ratio is 10:4;
S3, enzymolysis treatment: adding 100g of acellular dermal matrix into 500mL of water, adding 4g of alpha-galactosidase and 2.5g of trypsin, heating to 37 ℃, carrying out enzymolysis for 3 hours, filtering, washing and drying to obtain the acellular dermal matrix;
s4, preparation of hyaluronic acid/antibacterial peptide complex: adding 10g of hyaluronic acid into 200mL of water, adding 5gEDC g of NHS and 2.5g of NHS, performing activation reaction at 22 ℃ for 45min, adding 6g of antibacterial peptide, stirring and reacting for 13h, dialyzing, and freeze-drying to obtain hyaluronic acid/antibacterial peptide compound;
S5, preparing a decellularized matrix material: 3g of ferulic acid and 13g of antigen-free acellular dermal matrix are mixed and added into 200mL of water, 7g of EDC and 3.5g of NHS are added, activation reaction is carried out for 40min at 2 ℃, 8g of hyaluronic acid/antibacterial peptide complex is added, stirring reaction is carried out for 22h, dialysis and freeze drying are carried out, and the acellular matrix material is prepared.
Comparative example 2
In comparison with example 3, the difference is that no α -galactosidase was added in step S3.
The method comprises the following steps:
s1, pretreatment of pigskin: cleaning pigskin, dehairing, cleaning, removing epidermis cuticle and subcutaneous fat layer, taking dermis layer, cutting into proper size, soaking with 3% sodium hydroxide solution, and oscillating for 5h to obtain pretreated pigskin material;
s2, cell removal treatment: adding the pretreated pigskin material into ionic liquid, wherein the solid-to-liquid ratio of the pretreated pigskin material to the ionic liquid is 1:8g/mL, introducing carbon dioxide, and the ventilation amount is 25mL/min, stirring for 1.5h, dialyzing, and freeze-drying to obtain a acellular dermal matrix;
The ionic liquid is a mixture of 1-propyl-3-methylimidazole hexafluorophosphate and 1-butyl-2, 3-dimethylimidazole trifluoromethane sulfonate, and the mass ratio is 10:4;
s3, enzymolysis treatment: adding 100g of acellular dermal matrix into 500mL of water, adding 6.5g of trypsin, heating to 37 ℃, carrying out enzymolysis for 3 hours, filtering, washing and drying to obtain the acellular dermal matrix;
s4, preparation of hyaluronic acid/antibacterial peptide complex: adding 10g of hyaluronic acid into 200mL of water, adding 5gEDC g of NHS and 2.5g of NHS, performing activation reaction at 22 ℃ for 45min, adding 6g of antibacterial peptide, stirring and reacting for 13h, dialyzing, and freeze-drying to obtain hyaluronic acid/antibacterial peptide compound;
S5, preparing a decellularized matrix material: 3g of ferulic acid and 13g of antigen-free acellular dermal matrix are mixed and added into 200mL of water, 7g of EDC and 3.5g of NHS are added, activation reaction is carried out for 40min at 2 ℃, 8g of hyaluronic acid/antibacterial peptide complex is added, stirring reaction is carried out for 22h, dialysis and freeze drying are carried out, and the acellular matrix material is prepared.
Comparative example 3
In comparison with example 3, the difference is that no trypsin was added in step S3.
The method comprises the following steps:
s1, pretreatment of pigskin: cleaning pigskin, dehairing, cleaning, removing epidermis cuticle and subcutaneous fat layer, taking dermis layer, cutting into proper size, soaking with 3% sodium hydroxide solution, and oscillating for 5h to obtain pretreated pigskin material;
s2, cell removal treatment: adding the pretreated pigskin material into ionic liquid, wherein the solid-to-liquid ratio of the pretreated pigskin material to the ionic liquid is 1:8g/mL, introducing carbon dioxide, and the ventilation amount is 25mL/min, stirring for 1.5h, dialyzing, and freeze-drying to obtain a acellular dermal matrix;
The ionic liquid is a mixture of 1-propyl-3-methylimidazole hexafluorophosphate and 1-butyl-2, 3-dimethylimidazole trifluoromethane sulfonate, and the mass ratio is 10:4;
s3, enzymolysis treatment: adding 100g of acellular dermal matrix into 500mL of water, adding 6.5g of alpha-galactosidase, heating to 37 ℃, carrying out enzymolysis for 3h, filtering, washing and drying to obtain the acellular dermal matrix;
s4, preparation of hyaluronic acid/antibacterial peptide complex: adding 10g of hyaluronic acid into 200mL of water, adding 5gEDC g of NHS and 2.5g of NHS, performing activation reaction at 22 ℃ for 45min, adding 6g of antibacterial peptide, stirring and reacting for 13h, dialyzing, and freeze-drying to obtain hyaluronic acid/antibacterial peptide compound;
S5, preparing a decellularized matrix material: 3g of ferulic acid and 13g of antigen-free acellular dermal matrix are mixed and added into 200mL of water, 7g of EDC and 3.5g of NHS are added, activation reaction is carried out for 40min at 2 ℃, 8g of hyaluronic acid/antibacterial peptide complex is added, stirring reaction is carried out for 22h, dialysis and freeze drying are carried out, and the acellular matrix material is prepared.
Comparative example 4
In comparison with example 3, the difference is that step S3 is not performed.
The method comprises the following steps:
s1, pretreatment of pigskin: cleaning pigskin, dehairing, cleaning, removing epidermis cuticle and subcutaneous fat layer, taking dermis layer, cutting into proper size, soaking with 3% sodium hydroxide solution, and oscillating for 5h to obtain pretreated pigskin material;
s2, cell removal treatment: adding the pretreated pigskin material into ionic liquid, wherein the solid-to-liquid ratio of the pretreated pigskin material to the ionic liquid is 1:8g/mL, introducing carbon dioxide, and the ventilation amount is 25mL/min, stirring for 1.5h, dialyzing, and freeze-drying to obtain a acellular dermal matrix;
The ionic liquid is a mixture of 1-propyl-3-methylimidazole hexafluorophosphate and 1-butyl-2, 3-dimethylimidazole trifluoromethane sulfonate, and the mass ratio is 10:4;
S3, preparing a hyaluronic acid/antibacterial peptide compound: adding 10g of hyaluronic acid into 200mL of water, adding 5gEDC g of NHS and 2.5g of NHS, performing activation reaction at 22 ℃ for 45min, adding 6g of antibacterial peptide, stirring and reacting for 13h, dialyzing, and freeze-drying to obtain hyaluronic acid/antibacterial peptide compound;
S4, preparing a decellularized matrix material: 3g of ferulic acid and 13g of acellular dermal matrix are mixed and added into 200mL of water, 7g of EDC and 3.5g of NHS are added, activation reaction is carried out for 40min at 2 ℃, 8g of hyaluronic acid/antibacterial peptide complex is added, stirring reaction is carried out for 22h, dialysis and freeze drying are carried out, and acellular matrix material is prepared.
Comparative example 5
The difference compared to example 3 is that hyaluronic acid/antimicrobial peptide complex is not added in step S5.
The method comprises the following steps:
s1, pretreatment of pigskin: cleaning pigskin, dehairing, cleaning, removing epidermis cuticle and subcutaneous fat layer, taking dermis layer, cutting into proper size, soaking with 3% sodium hydroxide solution, and oscillating for 5h to obtain pretreated pigskin material;
s2, cell removal treatment: adding the pretreated pigskin material into ionic liquid, wherein the solid-to-liquid ratio of the pretreated pigskin material to the ionic liquid is 1:8g/mL, introducing carbon dioxide, and the ventilation amount is 25mL/min, stirring for 1.5h, dialyzing, and freeze-drying to obtain a acellular dermal matrix;
The ionic liquid is a mixture of 1-propyl-3-methylimidazole hexafluorophosphate and 1-butyl-2, 3-dimethylimidazole trifluoromethane sulfonate, and the mass ratio is 10:4;
S3, enzymolysis treatment: adding 100g of acellular dermal matrix into 500mL of water, adding 4g of alpha-galactosidase and 2.5g of trypsin, heating to 37 ℃, carrying out enzymolysis for 3 hours, filtering, washing and drying to obtain the acellular dermal matrix;
s4, preparation of hyaluronic acid/antibacterial peptide complex: adding 10g of hyaluronic acid into 200mL of water, adding 5gEDC g of NHS and 2.5g of NHS, performing activation reaction at 22 ℃ for 45min, adding 6g of antibacterial peptide, stirring and reacting for 13h, dialyzing, and freeze-drying to obtain hyaluronic acid/antibacterial peptide compound;
S5, preparing a decellularized matrix material: 11g of ferulic acid is mixed and added into 200mL of water, 7gEDC g of NHS and 3.5g of NHS are added, activation reaction is carried out for 40min at 2 ℃, 13g of antigen-removing acellular dermal matrix is added, stirring reaction is carried out for 22h, dialysis and freeze drying are carried out, and the acellular matrix material is prepared.
Comparative example 6
In comparison with example 3, the difference is that no ferulic acid was added in step S5.
The method comprises the following steps:
s1, pretreatment of pigskin: cleaning pigskin, dehairing, cleaning, removing epidermis cuticle and subcutaneous fat layer, taking dermis layer, cutting into proper size, soaking with 3% sodium hydroxide solution, and oscillating for 5h to obtain pretreated pigskin material;
s2, cell removal treatment: adding the pretreated pigskin material into ionic liquid, wherein the solid-to-liquid ratio of the pretreated pigskin material to the ionic liquid is 1:8g/mL, introducing carbon dioxide, and the ventilation amount is 25mL/min, stirring for 1.5h, dialyzing, and freeze-drying to obtain a acellular dermal matrix;
The ionic liquid is a mixture of 1-propyl-3-methylimidazole hexafluorophosphate and 1-butyl-2, 3-dimethylimidazole trifluoromethane sulfonate, and the mass ratio is 10:4;
S3, enzymolysis treatment: adding 100g of acellular dermal matrix into 500mL of water, adding 4g of alpha-galactosidase and 2.5g of trypsin, heating to 37 ℃, carrying out enzymolysis for 3 hours, filtering, washing and drying to obtain the acellular dermal matrix;
s4, preparation of hyaluronic acid/antibacterial peptide complex: adding 10g of hyaluronic acid into 200mL of water, adding 5gEDC g of NHS and 2.5g of NHS, performing activation reaction at 22 ℃ for 45min, adding 6g of antibacterial peptide, stirring and reacting for 13h, dialyzing, and freeze-drying to obtain hyaluronic acid/antibacterial peptide compound;
S5, preparing a decellularized matrix material: 13g of antigen-free decellularized dermal matrix is mixed and added into 200mL of water, 7g of EDC and 3.5g of NHS are added, activation reaction is carried out for 40min at 2 ℃, 11g of hyaluronic acid/antibacterial peptide complex is added, stirring reaction is carried out for 22h, dialysis and freeze drying are carried out, and thus the decellularized matrix material is prepared.
Comparative example 7
The difference compared to example 3 is that NHS and EDC are not added in step S5.
The method comprises the following steps:
s1, pretreatment of pigskin: cleaning pigskin, dehairing, cleaning, removing epidermis cuticle and subcutaneous fat layer, taking dermis layer, cutting into proper size, soaking with 3% sodium hydroxide solution, and oscillating for 5h to obtain pretreated pigskin material;
s2, cell removal treatment: adding the pretreated pigskin material into ionic liquid, wherein the solid-to-liquid ratio of the pretreated pigskin material to the ionic liquid is 1:8g/mL, introducing carbon dioxide, and the ventilation amount is 25mL/min, stirring for 1.5h, dialyzing, and freeze-drying to obtain a acellular dermal matrix;
The ionic liquid is a mixture of 1-propyl-3-methylimidazole hexafluorophosphate and 1-butyl-2, 3-dimethylimidazole trifluoromethane sulfonate, and the mass ratio is 10:4;
S3, enzymolysis treatment: adding 100g of acellular dermal matrix into 500mL of water, adding 4g of alpha-galactosidase and 2.5g of trypsin, heating to 37 ℃, carrying out enzymolysis for 3 hours, filtering, washing and drying to obtain the acellular dermal matrix;
s4, preparation of hyaluronic acid/antibacterial peptide complex: adding 10g of hyaluronic acid into 200mL of water, adding 5gEDC g of NHS and 2.5g of NHS, performing activation reaction at 22 ℃ for 45min, adding 6g of antibacterial peptide, stirring and reacting for 13h, dialyzing, and freeze-drying to obtain hyaluronic acid/antibacterial peptide compound;
s5, preparing a decellularized matrix material: mixing 3g of ferulic acid and 13g of antigen-free acellular dermal matrix, adding into 200mL of water, adding 8g of hyaluronic acid/antibacterial peptide complex, stirring for reacting for 22h, dialyzing, and freeze-drying to obtain the acellular matrix material.
Test example 1CCK-8 eukaryotic cytotoxicity assay
The decellularized matrix material solutions prepared in examples 1-3 or comparative examples 1-7 were formulated with deionized water at 0.5 wt%.
HaCaT or NIH-3T3 cells were digested, centrifuged, counted, inoculated into 96-well plates at a density of 1X 104/well, incubated at 37℃until the cell confluency was 70% -80%, 200. Mu.L of serum-free DMEM (MEM medium for HaCaT cells) was added to the tube, 10. Mu.L of a decellularized matrix material solution was added, incubated at 37℃for 2 hours, DMEM containing 20% fetal bovine serum was added, the mixture was changed to 96-well plates after mixing, incubation was continued for 24 hours, CCK-8 solution was added, incubation at 37℃for 1.5 hours, the D450 nm value was measured, and relative cell viability (%) was calculated using the blank (deionized water) as a control group. The results are shown in Table 1.
TABLE 1
| Group of | HaCaT cell relative cell viability (%) | NIH-3T3 cell relative cell viability (%) |
| Blank group | 100 | 100 |
| Example 1 | 102 | 104 |
| Example 2 | 104 | 105 |
| Example 3 | 105 | 107 |
| Comparative example 1 | 101 | 102 |
| Comparative example 2 | 97 | 99 |
| Comparative example 3 | 100 | 101 |
| Comparative example 4 | 94 | 96 |
| Comparative example 5 | 98 | 96 |
| Comparative example 6 | 95 | 97 |
| Comparative example 7 | 99 | 101 |
As can be seen from the above table, the cell-free matrix materials prepared in examples 1-3 of the present invention were very low in cytotoxicity.
Test example 2 type I collagenase degradation experiment
Putting the acellular matrix materials prepared in examples 1-3 or comparative examples 1-7 into a centrifuge tube, proportionally adding 1mg/mL of type I collagenase (purchased from Centipede Biotechnology Co., ltd.), putting into a constant temperature culture oscillator for reaction (37+/-0.1 ℃,120 r/min), taking out samples respectively at 12 hours, 24 hours and 48 hours (5 samples at each sampling point), drying, weighing, calculating the degradation rate, and simultaneously detecting the content of hydroxyproline in the degradation liquid at the sampling time point and observing the appearance turbidity degree of the degradation liquid.
Degradation rate = mass before degradation-mass after degradation x 100% mass before degradation
The results are shown in Table 2.
TABLE 2
As can be seen from the above table, the acellular matrix materials prepared in examples 1-3 of the present invention were degradable and the degradation rate was appropriate.
Test example 3 mechanical Property detection
The decellularized matrix material prepared in examples 1 to 3 or comparative examples 1 to 7 was cut into 50mm×4mm rectangles, and the tensile strength and elongation at break of the samples were examined. The results are shown in Table 3.
TABLE 3 Table 3
As can be seen from the above table, the acellular matrix materials prepared in examples 1-3 of the present invention have good mechanical properties.
Test example 4 anti-inflammatory experiment
Preparation of sample solution: the acellular matrix material solution was prepared with 0.36wt% NaCl solution at a concentration of 0.5wt%. The blood of the rats was centrifuged at 4000r/min for 15min, and the obtained erythrocytes were washed with 0.85% sodium chloride solution (ph=7.2) to prepare erythrocyte suspension (10% v/v), and the anti-inflammatory activity was measured. Distilled water was used as a control, and the mixture was incubated with 2mL of the sample solution, 0.15mol/L of phosphate buffer (pH=7.4), 1mL of 0.15mol/L and 0.5mL of the erythrocyte suspension, respectively, for 30min at 37℃and then centrifuged at 4000r/min for 15min. The supernatant was taken and assayed for hemoglobin by 560nm spectrophotometry. Erythrocyte membrane stability (%):
Erythrocyte membrane stability (%) = (1-a Sample of/A control) ×100%
Diclofenac at equal concentration was used as a positive control drug.
The results are shown in Table 4.
TABLE 4 Table 4
Diclofenac is an anti-inflammatory drug, and can be used as a reference of positive control, and the table shows that the acellular matrix materials prepared in the embodiments 1-3 have better anti-inflammatory performance.
Test example 5 determination of cellular NO content
After the macrophage RAW264.7 cells in the logarithmic growth phase were counted, the cell concentration was adjusted to 2X 105 cells/mL. Cells were plated in 6-well plates at 2mL per well, decellularized matrix material (0.5 mg/L) prepared in examples 1 to 3 or comparative examples 1 to 7 was added after the cells had adhered to the walls, LPS (0.5. Mu.g/L) was allowed to act at 37℃for 24 hours under 5% CO2, 50. Mu.L of each group of cell supernatants was collected, added to 96-well plates at 50. Mu.L per well, then GRIESSREAGENT I50. Mu.L was added, reacted in an incubator at 37℃for 10 minutes, then GRIESSREAGENT II 50. Mu.L was added to each well, and after shaking and mixing were performed uniformly, absorbance values of each group were measured at 540nm wavelength after light-shielding reaction at 37 ℃. Control wells were deionized water instead of supernatant. The blank group is without acellular matrix material and LPS added, and the positive group is dexamethasone instead of acellular matrix material.
NO content= [1- (a Drug delivery hole-A control wells)/A control wells) ] ×100%
The results are shown in Table 5.
TABLE 5
As shown in the table above, the acellular matrix materials prepared in examples 1-3 of the present invention have a good effect of inhibiting the secretion of inflammatory factors by macrophages.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.