CN110917403A - Tissue engineering skin scaffold material and preparation method thereof - Google Patents
Tissue engineering skin scaffold material and preparation method thereof Download PDFInfo
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- CN110917403A CN110917403A CN201911259473.2A CN201911259473A CN110917403A CN 110917403 A CN110917403 A CN 110917403A CN 201911259473 A CN201911259473 A CN 201911259473A CN 110917403 A CN110917403 A CN 110917403A
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- hyaluronic acid
- liposome
- superoxide dismutase
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Abstract
The invention provides a tissue engineering skin scaffold material, which consists of a hyaluronic acid scaffold and a nanofiber membrane; the hyaluronic acid scaffold is prepared by loading superoxide dismutase liposome on a hyaluronic acid porous scaffold; the nanofiber membrane is prepared from a chitosan/keratin mixed solution. The invention also provides a preparation method of the tissue engineering skin scaffold material, the double-layer skin scaffold material prepared by adopting the method of combining the electrostatic spinning technology and the freeze drying technology solves the problems of poor biocompatibility and improper pore size of the skin scaffold material and the problem that wound skin is influenced by harmful metabolites, and the preparation method is simple, green and environment-friendly.
Description
Technical Field
The invention belongs to the field of biological scaffold materials, and particularly relates to a tissue engineering skin scaffold material and a preparation method thereof.
Background
The skin is the largest organ of the human body, covers the whole body, and protects various tissues and organs in the body from physical, mechanical, chemical and pathogenic microorganisms, so when the skin is subjected to external attack, the skin is often firstly damaged, for example, skin injury caused by burns, scalds or diseases, and the like, and currently, the skin tissue injury of the human body is mostly treated by an autologous or allogeneic skin transplantation method, but the treatment method is not only limited in material source, but also can cause secondary damage to patients. With the development of medical science, the tissue engineering skin scaffold material provides a suitable environment for the in vitro culture of skin cells, and the success of the clinical application of the tissue engineering skin brings hope to patients with skin injury.
However, current materials still contain some drawbacks, such as: poor biocompatibility and improper pore diameter of the stent material, so that the degradable biological material is required to be used as a skin substitute with high quality and high applicability, the material per se has no immunogenicity to tissues, and the degraded product of the material has no toxicity or abnormal reaction to biological tissues; secondly, the prepared skin stent material has excellent mechanical property, has a pore size suitable for cell growth, is easy to induce the generation of capillaries and blood vessels, and does not change the shape and the size of the capillaries and the blood vessels after growing into the pore size; finally, the growth of the bacteria can be inhibited and growth factors that promote cell development can be released.
The body can release some harmful substances in the process of metabolism, sweat glands contained in normal skin can promote the excretion of the harmful substances, for skin injury patients, the sweat glands of mild injury patients can be slowly repaired, the sweat glands of severe patients can not be regenerated, the body can not smoothly excrete toxins or the harmful substances, the quantity of the harmful substances is gradually increased, the skin repair is inhibited, and the application of the skin bracket material can be further promoted by effectively solving the problem. Superoxide dismutase is an active substance commonly existing in animals, plants and microorganisms, can eliminate harmful substances such as superoxide radical generated in the metabolism process of organisms, has super-strong antioxidant and anti-inflammatory functions, can quickly recover damaged cell tissues and enable the damaged cell tissues to quickly heal, but has short active half-life after the superoxide dismutase enters cells, so that the effect of the superoxide dismutase is influenced, and therefore, the problem is also very important to solve.
Disclosure of Invention
The invention aims to provide a tissue engineering skin scaffold material and a preparation method thereof aiming at the defects of the prior art, and the invention adopts a double-layer skin scaffold material prepared by combining an electrostatic spinning technology and a freeze drying technology, thereby solving the problems that the wound skin is influenced by harmful metabolites and the scaffold material has poor biocompatibility and improper pore size.
In order to solve the technical problems, the invention adopts the technical scheme that:
a tissue engineering skin scaffold material consists of a hyaluronic acid scaffold and a nanofiber membrane; the hyaluronic acid scaffold is prepared by loading superoxide dismutase liposome on a hyaluronic acid porous scaffold; the nano-fiber membrane is prepared from a chitosan/keratin solution, the thickness of the hyaluronic acid scaffold is 1.2-5.0 mm, and the pore size is 60-100 mu m; the thickness of the nanofiber membrane is 0.04-0.8 mm, and the pore size is 10-40 mu m;
the preparation method of the superoxide dismutase liposome comprises the following steps: dissolving superoxide dismutase in water, mixing ether solution of sphingomyelin and cholesterol with superoxide dismutase aqueous solution, oscillating to form emulsion, then decompressing and evaporating to remove ether to obtain gel-state substances, adding phosphate buffer solution with pH of 7.0, oscillating to make gel fall off, and continuing decompressing and evaporating to obtain superoxide dismutase liposome, wherein the ratio of sphingomyelin to cholesterol is 1: 5-8, the concentration of the ether solution of sphingomyelin and cholesterol is 20%, the concentration of the superoxide dismutase aqueous solution is 2%, the volume ratio of the ether solution of phospholipid to the superoxide dismutase aqueous solution is 1: 1-1: 1.5, and the encapsulation rate of the liposome reaches 86-91%.
A preparation method of a tissue engineering skin scaffold material specifically comprises the following steps:
dissolving chitosan and keratin in a weak acid solution according to a mass ratio of 2: 1-10: 1, and uniformly stirring to obtain a chitosan/keratin solution; dissolving hyaluronic acid in ultrapure water to obtain a hyaluronic acid solution;
step two, uniformly mixing the hyaluronic acid solution prepared in the step one with a sodium acetate solution, casting the mixture into a mold, freezing and freeze-drying the mixture at a temperature of between 15 ℃ below zero and 75 ℃ below zero to obtain a hyaluronic acid porous scaffold containing sodium acetate, putting the hyaluronic acid porous scaffold into an aseptic tray, pouring the liposome of superoxide dismutase on the hyaluronic acid porous scaffold to ensure that the scaffold is completely immersed in the solution, ultrasonically oscillating the mixture for half an hour to load the liposome on the porous scaffold, taking out the hyaluronic acid scaffold loaded with the liposome, and washing the hyaluronic acid scaffold with ultrapure water to remove the liposome solution remaining on the surface for later use;
step three, putting the chitosan/keratin solution prepared in the step one into a needle tube of electrostatic spinning equipment, fixing the hyaluronic acid bracket loaded with the liposome prepared in the step two on an aluminum foil of a receiving device of the electrostatic spinning equipment, grounding the aluminum foil, pushing the needle tube by high-voltage static electricity to enable the chitosan/keratin solution to form nano fibers attached to the surface of a porous bracket, and forming a layer of nano fiber film on the surface of the hyaluronic acid bracket as a surface layer bracket after a certain time to obtain a double-layer bracket material;
and step four, soaking the prepared double-layer scaffold in water, repeatedly washing the scaffold with a large amount of ultrapure water to remove residual formic acid, acetic acid and sodium acetate on the scaffold, and then drying the scaffold at a constant temperature (25 ℃) in vacuum to obtain the tissue engineering skin scaffold material.
Preferably, in the step one, the weak acid is a mixed solution of formic acid and acetic acid (1: 1-5: 1), and the concentration of the chitosan/keratin solution is 6-9%; the concentration of the hyaluronic acid solution is 12-18%.
Preferably, the concentration of the sodium acetate solution in the second step is 4.0-6.0%, and the volume ratio of the hyaluronic acid solution to the sodium acetate solution is 1: 1-2: 1.
Preferably, the thickness of the mixed solution of hyaluronic acid and sodium acetate cast in the mold in the second step is 1.2-4.8 mm, and the ultrasonic oscillation is performed by using an ultrasonic oscillator with the temperature of 10-20 ℃ and the power of 20-40W.
Preferably, the spinning voltage in the third step is 20-30 kv, the distance between the needle tube and the receiving device is 10-20 cm, and the filament discharging speed of the nanofiber filament is 0.5-1 mL/h.
Preferably, the porosity of the nanofiber membrane in the third step is 91-94%.
The dermal layer bracket prepared by loading the superoxide dismutase liposome on the hyaluronic acid bracket can promote the growth of granulation tissues, accelerate the healing speed of wound surfaces, reduce inflammatory reaction and eliminate harmful substances generated by organisms in the metabolism process; the superoxide dismutase is wrapped by the liposome to ensure the activity of the superoxide dismutase, and the superoxide dismutase is slowly released to prolong the action period of the superoxide dismutase; the nano fibrous membrane epidermal layer bracket is prepared by utilizing the chitosan/keratin solution, so that the possibility of immunological rejection of an organism to the nano fibrous membrane epidermal layer bracket is reduced, the mechanical property and porosity of a skin bracket material are increased, a growth factor released in the keratin degradation process can stimulate cells to secrete a large amount of collagen, elastic fibers and other substances, and the keratin can also induce macrophages to release fibroblast growth factors in the degradation process, promote mitosis of fibroblasts and stimulate blood vessel formation, finally heal a wound surface and repair skin.
Compared with the prior art, the invention has the following advantages:
(1) the invention leads the liposome to wrap the superoxide dismutase and load the superoxide dismutase on the hyaluronic acid porous bracket, can continuously release the superoxide dismutase, eliminate harmful substances generated by the metabolism of organisms, simultaneously promote the growth of granulation tissues and improve the utilization efficiency of the superoxide dismutase.
(2) The tissue engineering skin scaffold material prepared by the invention is a double-layer scaffold material, has excellent biocompatibility, and can release growth factors in the material degradation process, so that the epidermal layer and the body skin are fully integrated, the growth and survival of the dermal layer are ensured, and the recovery of the wound skin is promoted.
(3) The tissue engineering skin scaffold material is prepared by a method combining an electrostatic spinning technology and a freeze drying technology, the aperture of the porous scaffold is 60-100 mu m and is suitable for the growth of dermal fibroblasts, and the aperture of the nanofiber membrane is 10-40 mu m and is suitable for the growth of epidermal cells, so that the prepared skin scaffold material can be more fully integrated with wound surfaces.
(4) The tissue engineering skin scaffold material has good biocompatibility and can be biodegraded, the degraded product has no toxicity or abnormal reaction to biological tissues, and the preparation method is simple, green and environment-friendly.
Drawings
FIG. 1 is a graph showing the effect of different tissue engineering skin scaffold materials on fibroblast proliferation over time.
Detailed Description
The technical solutions of the present invention are further described in detail with reference to specific examples so that those skilled in the art can better understand the present invention and can implement the present invention, but the examples are not intended to limit the present invention.
Example 1
A tissue engineering skin scaffold material consists of a hyaluronic acid scaffold and a nanofiber membrane; the hyaluronic acid scaffold is prepared by loading superoxide dismutase liposome on a hyaluronic acid porous scaffold; the nano-fiber membrane is prepared from a chitosan/keratin solution, the thickness of the hyaluronic acid scaffold is 1.5mm, and the pore size is 83 microns; the thickness of the nanofiber membrane is 0.1mm, and the pore size is 35 mu m;
the preparation method of the superoxide dismutase liposome comprises the following steps: dissolving superoxide dismutase in water, mixing ether solution of sphingomyelin and cholesterol with superoxide dismutase aqueous solution, shaking to form emulsion, then removing ether by reduced pressure evaporation to obtain gel-state substance, adding phosphate buffer solution with pH of 7.0, shaking to make gel fall off, continuing to evaporate under reduced pressure to obtain superoxide dismutase liposome, wherein the ratio of sphingomyelin to cholesterol is 1:5, the concentration of the ether solution of sphingomyelin and cholesterol is 20%, the concentration of the superoxide dismutase aqueous solution is 2%, the volume ratio of the ether solution of phospholipid to the superoxide dismutase aqueous solution is 1:1, and the encapsulation rate of the liposome is 88%.
The preparation method comprises the following steps:
step one, dissolving 0.6g of chitosan and keratin in 10ml of weak acid solution according to the mass ratio of 2:1, and uniformly stirring to obtain a chitosan/keratin solution; dissolving 1.2g of hyaluronic acid in 10ml of ultrapure water to obtain a hyaluronic acid solution;
step two, uniformly mixing the hyaluronic acid solution prepared in the step one with a sodium acetate solution, casting the mixture into a mold, freezing and freeze-drying the mixture at a temperature of between 15 ℃ below zero and 75 ℃ below zero to obtain a hyaluronic acid porous scaffold containing sodium acetate, putting the obtained hyaluronic acid porous scaffold into an aseptic tray, pouring superoxide dismutase liposome on the hyaluronic acid porous scaffold to ensure that the scaffold is completely immersed in the solution, carrying the liposome on the porous scaffold by ultrasonic oscillation for half an hour, taking out the hyaluronic acid scaffold carrying the liposome, and washing the hyaluronic acid scaffold with ultrapure water to remove the liposome solution remaining on the surface for later use;
step three, putting the chitosan/keratin solution prepared in the step one into a needle tube of electrostatic spinning equipment, fixing the hyaluronic acid bracket loaded with the liposome prepared in the step two on an aluminum foil of a receiving device of the electrostatic spinning equipment, grounding the aluminum foil, pushing the needle tube by high-voltage static electricity to enable the chitosan/keratin solution to form nano fibers attached to the surface of a porous bracket, and forming a layer of nano fiber film on the surface of the hyaluronic acid bracket as a surface layer bracket after a certain time to obtain a double-layer bracket material;
and step four, soaking the prepared double-layer scaffold in water, repeatedly washing the scaffold with a large amount of ultrapure water to remove residual formic acid, acetic acid and sodium acetate on the scaffold, and then drying the scaffold at a constant temperature (25 ℃) in vacuum to obtain the tissue engineering skin scaffold material.
In the first step, the weak acid is a mixed solution of formic acid and acetic acid (1:1), and the concentration of the chitosan/keratin solution is 6 percent; the concentration of the hyaluronic acid solution was 12%.
In the second step, the concentration of the sodium acetate solution is 4.0%, and the volume ratio of the hyaluronic acid solution to the sodium acetate solution is 2: 1.
And in the second step, the thickness of the mixed solution of the hyaluronic acid and the sodium acetate cast in the mould is 1.5mm, and the ultrasonic oscillation is carried out by adopting an ultrasonic oscillator with the temperature of 10 ℃ and the power of 20W.
In the third step, the spinning voltage is 15kv, the distance between the needle tube and the receiving device is 10cm, and the filament discharging speed of the nano-fiber filament is 0.5 mL/h.
The porosity of the nanofiber membrane in the third step reaches 93 percent.
Example 2
A tissue engineering skin scaffold material consists of a hyaluronic acid scaffold and a nanofiber membrane; the hyaluronic acid scaffold is prepared by loading superoxide dismutase liposome on a hyaluronic acid porous scaffold; the nano-fiber membrane is prepared from a chitosan/keratin solution, the thickness of the hyaluronic acid scaffold is 2.3mm, and the pore size is 96 mu m; the thickness of the nanofiber membrane is 0.35mm, and the pore size is 25 micrometers;
the preparation method of the superoxide dismutase liposome comprises the following steps: dissolving superoxide dismutase in water, mixing ether solution of sphingomyelin and cholesterol with superoxide dismutase aqueous solution, shaking to form emulsion, then removing ether by reduced pressure evaporation to obtain gel-state substance, adding phosphate buffer solution with pH of 7.0, shaking to make gel fall off, continuing to evaporate under reduced pressure to obtain superoxide dismutase liposome, wherein the ratio of sphingomyelin to cholesterol is 1:6, the concentration of the ether solution of sphingomyelin and cholesterol is 20%, the concentration of the superoxide dismutase aqueous solution is 2%, the volume ratio of the ether solution of phospholipid to the superoxide dismutase aqueous solution is 1:1.2, and the encapsulation rate of the liposome reaches 91%.
The preparation method comprises the following steps:
step one, dissolving 0.8g of chitosan and keratin in 10ml of weak acid solution according to the mass ratio of 6:1, and uniformly stirring to obtain a chitosan/keratin solution; dissolving 1.5g of hyaluronic acid in 10ml of ultrapure water to obtain a hyaluronic acid solution;
step two, uniformly mixing the hyaluronic acid solution prepared in the step one with a sodium acetate solution, casting the mixture into a mold, freezing and freeze-drying the mixture at a temperature of between 15 ℃ below zero and 75 ℃ below zero to obtain a hyaluronic acid porous scaffold containing sodium acetate, putting the obtained hyaluronic acid porous scaffold into an aseptic tray, pouring superoxide dismutase liposome on the hyaluronic acid porous scaffold to ensure that the scaffold is completely immersed in the solution, carrying the liposome on the porous scaffold by ultrasonic oscillation for half an hour, taking out the hyaluronic acid scaffold carrying the liposome, and washing the hyaluronic acid scaffold with ultrapure water to remove the liposome solution remaining on the surface for later use;
step three, putting the chitosan/keratin solution prepared in the step one into a needle tube of electrostatic spinning equipment, fixing the hyaluronic acid bracket loaded with the liposome prepared in the step two on an aluminum foil of a receiving device of the electrostatic spinning equipment, grounding the aluminum foil, pushing the needle tube by high-voltage static electricity to enable the chitosan/keratin solution to form nano fibers attached to the surface of a porous bracket, and forming a layer of nano fiber film on the surface of the hyaluronic acid bracket as a surface layer bracket after a certain time to obtain a double-layer bracket material;
and step four, soaking the prepared double-layer scaffold in water, repeatedly washing the scaffold with a large amount of ultrapure water to remove residual formic acid, acetic acid and sodium acetate on the scaffold, and then drying the scaffold at a constant temperature (25 ℃) in vacuum to obtain the tissue engineering skin scaffold material.
In the first step, the weak acid is a mixed solution of formic acid and acetic acid (3:1), and the concentration of the chitosan/keratin solution is 8 percent; the concentration of the hyaluronic acid solution was 15%.
In the second step, the concentration of the sodium acetate solution is 5.0%, and the volume ratio of the hyaluronic acid solution to the sodium acetate solution is 1:1.
And in the second step, the thickness of the mixed solution of the hyaluronic acid and the sodium acetate cast in the mould is 3.0mm, and the ultrasonic oscillation is carried out by adopting an ultrasonic oscillator with the temperature of 15 ℃ and the power of 30W.
In the third step, the spinning voltage is 15kv, the distance between the needle tube and the receiving device is 15cm, and the filament discharging speed of the nano-fiber filament is 1 mL/h.
The porosity of the nanofiber membrane in the third step reaches 91%.
Example 3
A tissue engineering skin scaffold material consists of a hyaluronic acid scaffold and a nanofiber membrane; the hyaluronic acid scaffold is prepared by loading superoxide dismutase liposome on a hyaluronic acid porous scaffold; the nano-fiber membrane is prepared from a chitosan/keratin solution, the thickness of the hyaluronic acid scaffold is 4.4mm, and the pore size is 70 mu m; the thickness of the nanofiber membrane is 0.6mm, and the pore size is 40 mu m;
the preparation method of the superoxide dismutase liposome comprises the following steps: dissolving superoxide dismutase in water, mixing ether solution of sphingomyelin and cholesterol with superoxide dismutase aqueous solution, shaking to form emulsion, then removing ether by reduced pressure evaporation to obtain gel-state substance, adding phosphate buffer solution with pH of 7.0, shaking to make gel fall off, continuing to evaporate under reduced pressure to obtain superoxide dismutase liposome, wherein the ratio of sphingomyelin to cholesterol is 1:8, the concentration of the ether solution of sphingomyelin and cholesterol is 20%, the concentration of the superoxide dismutase aqueous solution is 2%, the volume ratio of the ether solution of phospholipid to the superoxide dismutase aqueous solution is 1:1.5, and the encapsulation rate of the liposome reaches 86%.
The preparation method comprises the following steps:
step one, dissolving 0.9g of chitosan and keratin in 10ml of weak acid solution according to the mass ratio of 10:1, and uniformly stirring to obtain a chitosan/keratin solution; dissolving 1.8g of hyaluronic acid in 10ml of ultrapure water to obtain a hyaluronic acid solution;
step two, uniformly mixing the hyaluronic acid solution prepared in the step one with a sodium acetate solution, casting the mixture into a mold, freezing and freeze-drying the mixture at a temperature of between 15 ℃ below zero and 75 ℃ below zero to obtain a hyaluronic acid porous scaffold containing sodium acetate, putting the obtained hyaluronic acid porous scaffold into an aseptic tray, pouring superoxide dismutase liposome on the hyaluronic acid porous scaffold to ensure that the scaffold is completely immersed in the solution, carrying the liposome on the porous scaffold by ultrasonic oscillation for half an hour, taking out the hyaluronic acid scaffold carrying the liposome, and washing the hyaluronic acid scaffold with ultrapure water to remove the liposome solution remaining on the surface for later use;
step three, putting the chitosan/keratin solution prepared in the step one into a needle tube of electrostatic spinning equipment, fixing the hyaluronic acid bracket loaded with the liposome prepared in the step two on an aluminum foil of a receiving device of the electrostatic spinning equipment, grounding the aluminum foil, pushing the needle tube by high-voltage static electricity to enable the chitosan/keratin solution to form nano fibers attached to the surface of a porous bracket, and forming a layer of nano fiber film on the surface of the hyaluronic acid bracket as a surface layer bracket after a certain time to obtain a double-layer bracket material;
and step four, soaking the prepared double-layer scaffold in water, repeatedly washing the scaffold with a large amount of ultrapure water to remove residual formic acid, acetic acid and sodium acetate on the scaffold, and then drying the scaffold at a constant temperature (25 ℃) in vacuum to obtain the tissue engineering skin scaffold material.
In the first step, the weak acid is a mixed solution of formic acid and acetic acid (5:1), and the concentration of the chitosan/keratin solution is 9 percent; the concentration of the hyaluronic acid solution is 18%;
in the second step, the concentration of the sodium acetate solution is 6.0%, and the volume ratio of the hyaluronic acid solution to the sodium acetate solution is 1:1.
And in the second step, the thickness of the mixed solution of the hyaluronic acid and the sodium acetate cast in the mould is 4.5mm, and the ultrasonic oscillation is carried out by adopting an ultrasonic oscillator with the temperature of 20 ℃ and the power of 40W.
In the third step, the spinning voltage is 20kv, the distance between the needle tube and the receiving device is 20cm, and the filament discharging speed of the nano-fiber filament is 1 mL/h.
The porosity of the nanofiber membrane in the third step reaches 94%.
Comparative example 1
Compared with example 1, in comparative example 1, the superoxide dismutase is not contained, the dermal layer porous scaffold is made of hyaluronic acid only, and the rest of the composition ratio and the preparation method are the same as those of example 1.
Comparative example 2
Compared with the example 1, in the comparative example 2, the chitosan/keratin solution is used as the material for preparing the dermal layer porous scaffold, the hyaluronic acid solution and the liposome solution are mixed to be used as the material for preparing the epidermal layer nanofiber membrane, and the rest composition ratio and the preparation method are the same as those of the example 1.
Comparative example 3
Compared with the example 1, in the comparative example 3, the superoxide dismutase is directly mixed with the hyaluronic acid solution and the hyaluronic acid porous scaffold is prepared by the freeze-drying method, and the rest of the composition ratio and the preparation method are the same as the example 1.
The method for measuring the pore diameter of the tissue engineering skin scaffold comprises the following steps: cutting the film longitudinally by using a scalpel, and observing the film under an electron microscope.
Wherein, the determination of the porosity of the tissue engineering skin scaffold comprises the following steps: the side length or radius and height of the material are measured, the volume V of the stent is calculated, the dry stent material is weighed in the air to be M, the stent material is immersed in liquid paraffin (density ρ), negative pressure degassing is performed to saturate the stent material, the stent material is removed, the medium on the surface is wiped off, the weight of the medium is called M, and the porosity is (M-M)/ρ/V × 100%.
Wherein the encapsulation rate of the superoxide dismutase liposome is determined as follows: adding a certain amount of prepared liposome into an ultrafiltration centrifugal tube, centrifuging for half an hour, and collecting filtrate. Breaking the same amount of non-ultrafiltered liposome and the collected filtrate with demulsifier, diluting to the same times, and determining the concentration of the drug in the collected filtrate to be C1The non-ultrafiltered liposomes released the encapsulated drug and the drug concentration was determined to be C2If the encapsulation efficiency is equal to (C)2-C1)/C2×100％。
Cell proliferation assay: fibroblast cells were cultured at 1X 104/cm3The cells were inoculated on the corresponding skin scaffold materials prepared in example 1 and comparative examples 1 to 3, and placed in the corresponding cell culture solutions of twenty-four well plates, and a Control group (Control) was set for direct cell culture in the wells, with four duplicate wells per group. The cells were stained with MTT kit every day from the first day to the fifth day, 200. mu.L/well of MTT (5 mg/mL in PBS) was added, incubation was continued for 5 hours, the culture solution was removed, the residual sample in the wells was washed with PBS solution two to three times, 500. mu.L/well of DMSO was added, the mixture was allowed to stand for 10 hours, shaken for 0min, and the absorbance was measured at 490nm on an immunomicroplate reader (FIG. 1).
The animal experiment method comprises the following steps: the experiments were divided into 7 experimental groups, which were: group 1 was the treatment of example 1, group 2 was the treatment of example 2, group 3 was the treatment of example 3, group 4 was the treatment of comparative example 1, group 5 was the treatment of comparative example 2, group 6 was the treatment of comparative example 3 and control group 7, SD rats 28 were present, 4 rats per group, weighing 200g, half of the male and female, hair was removed from the portion to be treated on the back of the rat the day before the experiment, the rat was anesthetized and injected with tramadol injection to relieve pain, the skin on the back of the rat was scalded with constant temperature water of 100 ℃ through a glass cylinder to cause a III degree scald of 1.5cm × 1.5cm size, immediately thereafter, 1mL of lactated ringer's solution was injected to assist the recovery and repair was performed with the prepared artificial skin scaffold material, groups 1 to 3 implanted the skin scaffold materials prepared in examples 1 to 3, groups 4 to 6 were implanted with the skin scaffold materials prepared in comparative examples 1 to 3, respectively, the material was attached to the wound surface tightly, sterile vaseline gauze was covered on the surface of the graft, the graft was fixed to the surrounding skin with sutures to prevent displacement of the graft, and then the graft was bandaged with sterile bandage pressure, and group 7 was not treated for grafting, and the other treatments were the same as those of group 1. The body temperature and food intake of the SD rat were observed after surgery, the drug was changed for the first time on the fourth day, then the drug was changed every two days, and the wound repair, infection or bleeding, and the survival of the graft were observed (14 days and 35 days after the graft, the whole skin layer at the junction between the wound and the wound was taken and observed with a microscope), with the specific results shown in Table 1.
TABLE 1 wound surface shrinkage (%) at different stages of SD rats after operation
| Day 8 | Day 14 | Day 20 | Day 26 | Day 32 | |
| Group 1 | 2.42±0.31 | 5.63±0.52 | 13.96±1.69 | 21.25±4.02 | 28.58±5.71 |
| Group 2 | 2.56±0.29 | 5.82±0.50 | 14.54±1.56 | 21.88±4.63 | 30.63±5.88 |
| Group 3 | 2.38±0.46 | 5.58±0.60 | 14.13±1.98 | 20.06±4.72 | 29.26±6.08 |
| Group 4 | 3.51±0.44 | 6.09±0.49 | 20.37±2.18 | 30.64±3.05 | 41.05±3.84 |
| Group 5 | 4.28±0.63 | 8.51±0.81 | 24.53±2.09 | 37.19±3.67 | 45.61±3.50 |
| Group 6 | 2.88±0.23 | 7.63±0.47 | 19.77±3.23 | 28.67±2.92 | 38.38±3.27 |
| Group 7 | 31.14±1.35 | 48.08±1.87 | 69.29±3.21 | 84.84±2.55 | 94.46±2.91 |
The wound surface is observed and the data in table 1 show that the SD rats of groups 1, 2 and 3 can be epithelialized at the wound surface edge when the days 4 to 8 are reached, the skin bracket material is tightly attached to the wound surface, the wound surface is dry, the wound is free of allergy and infection, the shrinkage rate is about one third of the wound surface when the time goes to 32 days, no turbid liquid secretion seeps out from the wound surface, and the skin repaired after 14 days and 35 days after the observation is found: the SD rat wound surfaces of groups 1 to 3 have most granulation tissues and new capillaries formed in 14 days, have very little inflammatory cell infiltration, have the granulation tissues and the new capillaries formed basically and completely in 35 days, have a large amount of fibroblast hyperplasia, have no inflammatory cell infiltration and are reconstructed, and the tissue engineering skin scaffold materials prepared in the embodiments 1 to 3 have anti-infection and anti-inflammatory effects, so that the wound surfaces can heal faster and better.
The wound surface shrinkage of the SD rats in group 4 was significantly increased at 32 days, but the wound surface was kept dry, free from allergy and infection, and when skin repair was performed for 14 days and 35 days, it was found that, at 14 days, the amount of granulation tissue and new capillaries formed was less than that of the SD rats in group 1, and the SD rats in group 1 were regenerated at 14 days, and also had inflammatory cell infiltration, and at 35 days, it was still observed that the wound surface skin contained a small amount of inflammatory cells, but granulation tissue and new capillaries were substantially completely formed, which illustrates that the tissue engineering skin scaffold material of comparative example 1 containing no superoxide dismutase had effects in anti-inflammation, wound surface shrinkage inhibition and granulation tissue growth promotion in the skin scaffold material of example 1.
The wound shrinkage of the SD rat in the group 5 after the operation for 32 days is more obviously increased compared with that of the SD rat in the group 1, epithelization of the edge of the wound can be observed in 4-8 days, the wound is dry and has no infection and allergy symptoms, and the rates of growth of granulation tissue, formation of new capillaries, proliferation of fibroblasts and reconstruction of skin are slightly slower than those of the wound skin of the SD rat in the group 1 when the skin is repaired after the operation, which shows that the effect of repairing the skin wound by adopting the skin scaffold material in the comparative example 2 is inferior to that of the skin scaffold material in the example 1, and further shows that the dermal layer porous scaffold prepared by the hyaluronic acid scaffold loaded with the liposome is more favorable for the growth of fibroblasts and granulation tissue, and the epidermal layer nanofiber membrane prepared by the chitosan/keratin solution can further ensure the survival rate of cells in the dermal layer.
The shrinkage of the wound surface of the SD rat in the group 6 before 14 days after the operation is still similar to that of the SD rat in the group 1, and the increase rate of the shrinkage of the wound surface after 14 days is higher than that of the SD rat in the group 1, and finally (38.38 ± 3.27)%. The repaired skin observed for 14 days and 35 days shows that no inflammatory cells exist at 14 days, granulation tissues and new capillaries are mostly formed, a small amount of inflammatory cells infiltrate at 35 days, a large amount of fibroblasts proliferate, the skin is reconstructed, and only the growth rates of the granulation tissues and the new capillaries are not as high as those of the SD rat of the group 1, which indicates that superoxide dismutase in the skin scaffold material of the comparative example 3 is not wrapped by liposome and is inactivated in the skin, so that the skin scaffold of the comparative example 3 has anti-inflammation within a certain time after transplantation, and then the inflammatory cells infiltrate the wound again after inactivation, and meanwhile, the growth rates of the granulation tissues and the new capillaries are relatively reduced.
The SD rats of group 7 rapidly contracted the wound after operation, and the wound adhered to gauze, and had a small amount of turbid liquid secretion, and healed to a linear crust about 35 days, and it was found by observing the wound 14 days and 35 days after operation that there was a large amount of inflammatory cell infiltration, partial necrosis, exudation, and the wound covered with granulation tissue, no regenerative epidermis was formed, and finally the wound contracted to a linear shape.
The SD rats in groups 1 to 3 have recovered appetite and spirit after the second to three days of operation, and have no obvious change compared with normal SD rats, the SD rats in groups 4 to 6 have slowly recovered spirit after the sixth to seven days of operation, but still are not as active as normal rats, the SD rats in group 7 have anorexia and lassitude after the operation, the weight of the SD rats slightly decreases before 10 days, and slowly increases after 10 days.
The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (8)
1. The tissue engineering skin scaffold material is characterized by consisting of a hyaluronic acid scaffold and a nanofiber membrane; the hyaluronic acid scaffold is prepared by loading superoxide dismutase liposome on a hyaluronic acid porous scaffold; the nano-fiber membrane is prepared from a chitosan/keratin solution, the thickness of the hyaluronic acid scaffold is 1.2-5.0 mm, and the pore size is 60-100 mu m; the thickness of the nanofiber membrane is 0.04-0.8 mm, and the pore size is 10-40 mu m.
2. The tissue engineering skin scaffold material of claim 1, wherein the superoxide dismutase liposome is prepared by the method comprising: dissolving superoxide dismutase in water, mixing ether solution of sphingomyelin and cholesterol with superoxide dismutase aqueous solution, shaking to form an emulsion, then removing ether by reduced pressure evaporation to obtain a gel-state substance, adding phosphate buffer solution with pH of 7.0, shaking to make the gel fall off, and continuing to evaporate under reduced pressure to obtain superoxide dismutase liposome, wherein the ratio of sphingomyelin to cholesterol is 1: 5-8, the concentration of the ether solution of sphingomyelin and cholesterol is 20%, the concentration of the superoxide dismutase aqueous solution is 2%, the volume ratio of the ether solution of phospholipid to the superoxide dismutase aqueous solution is 1: 1-1: 1.5, and the encapsulation rate of the liposome is 86% -91%.
3. The preparation method of the tissue engineering skin scaffold material according to any one of claims 1-2, which is characterized by comprising the following steps:
dissolving chitosan and keratin in a weak acid solution according to a mass ratio of 2: 1-10: 1, and uniformly stirring to obtain a chitosan/keratin solution; dissolving hyaluronic acid in deionized water to obtain a hyaluronic acid solution;
step two, uniformly mixing the hyaluronic acid solution prepared in the step one with a sodium acetate solution, casting the mixture into a mold, freezing and freeze-drying the mixture at the temperature of minus 15 ℃ to minus 75 ℃ to obtain a hyaluronic acid porous scaffold containing sodium acetate, then placing the obtained hyaluronic acid porous scaffold into an aseptic tray, pouring a liposome solution wrapping superoxide dismutase on the hyaluronic acid porous scaffold to ensure that the scaffold is completely immersed in the solution, ultrasonically oscillating the mixture for half an hour to load the liposome on the porous scaffold, then taking out the hyaluronic acid scaffold loaded with the liposome, and washing the hyaluronic acid scaffold with ultrapure water to remove the liposome solution remaining on the surface for later use;
step three, putting the chitosan/keratin solution prepared in the step one into a needle tube of electrostatic spinning equipment, fixing the hyaluronic acid bracket loaded with the liposome prepared in the step three on an aluminum foil of a receiving device of the electrostatic spinning equipment, grounding the aluminum foil, pushing the needle tube by high-voltage static electricity to enable the chitosan/keratin solution to form nano fibers attached to the surface of a porous bracket, and forming a layer of nano fiber film on the surface of the hyaluronic acid bracket as a surface layer bracket after a certain time to obtain a double-layer bracket material;
and step four, soaking the prepared double-layer scaffold in water, repeatedly washing the scaffold with a large amount of ultrapure water to remove residual formic acid, acetic acid and sodium acetate on the scaffold, and then drying the scaffold at a constant temperature (25 ℃) in vacuum to obtain the tissue engineering skin scaffold material.
4. The preparation method of the tissue engineering skin scaffold material according to claim 3, wherein the weak acid in the first step is a mixed solution of formic acid and acetic acid (1: 1-5: 1), the concentration of the chitosan/keratin solution is 6% -9%, and the concentration of the hyaluronic acid solution is 12% -18%.
5. The preparation method of the tissue engineering skin scaffold material according to claim 3, wherein the concentration of the sodium acetate solution in the second step is 4.0-6.0%, and the volume ratio of the hyaluronic acid solution to the sodium acetate solution is 1: 1-2: 1.
6. The preparation method of the tissue engineering skin scaffold material according to claim 3, wherein the thickness of the mixed solution of hyaluronic acid and sodium acetate cast in the mold in the second step is 1.2-4.8 mm, and the ultrasonic oscillation is performed by using an ultrasonic oscillator with the temperature of 10-20 ℃ and the power of 20-40W.
7. The preparation method of the tissue engineering skin scaffold material according to claim 3, wherein the spinning voltage in the third step is 20-30 kv, the distance between the needle tube and the receiving device is 10-20 cm, and the filament discharging speed of the nano-fiber filament is 0.5-1 mL/h.
8. The preparation method of the tissue engineering skin scaffold material according to claim 3, wherein the porosity of the nanofiber membrane in step three is 91% -94%.
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