CN115887785A - Antibacterial artificial skin and preparation method thereof - Google Patents
Antibacterial artificial skin and preparation method thereof Download PDFInfo
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- CN115887785A CN115887785A CN202211652205.9A CN202211652205A CN115887785A CN 115887785 A CN115887785 A CN 115887785A CN 202211652205 A CN202211652205 A CN 202211652205A CN 115887785 A CN115887785 A CN 115887785A
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention provides an antibacterial artificial skin and a preparation method thereof, belonging to the technical field of artificial skin, wherein the antibacterial artificial skin comprises a lower dermis layer, an upper dermis layer, an absorbable epidermis layer and a non-absorbable epidermis layer which are sequentially arranged from bottom to top; the lower dermis layer is a spongy porous collagen layer; the upper layer of the dermis is a porous acellular matrix layer; the porous acellular matrix layer at least comprises a layer of porous acellular matrix membrane adsorbed with active substances; the active substance comprises epidermal growth factor and basic fibroblast growth factor; the absorbable epidermal layer is an antibacterial acellular matrix layer; the bacteriostatic acellular substrate comprises an acellular substrate film coated with nano mesoporous bioactive glass on the surface; the non-absorbable skin layer is a film layer. The artificial skin provided by the invention has continuous antibacterial activity, can prevent wound infection, can effectively promote the healing of soft tissue injury wound, improves the tissue regeneration and repair capacity, and accelerates the reconstruction of dermis.
Description
Technical Field
The invention belongs to the technical field of artificial skin, and particularly relates to antibacterial artificial skin and a preparation method thereof.
Background
The skin is the largest organ of a human body, and is damaged by trauma, burn, diabetic foot, tumor resection and the like; at present, the clinical repair method for deep or large-area skin defects is autologous skin flap or flap transplantation, but the autologous skin flap or flap transplantation can cause donor area damage; the problems of immunological rejection, use safety and the like exist in the transplantation of the allogeneic skin sheet or the skin flap.
The artificial skin can be used for treating wounds with damaged dermis, chronic wounds or burn wounds as a substitute for autologous skin transplantation, has the effects of promoting wound healing, reducing scar formation and the like, overcomes the defect of insufficient autologous transplantation skin resources of patients with large-area burns, and gradually occupies the mainstream of the market. However, the artificial skin fails to establish blood supply with the wound surface in the early stage after transplantation, the antibacterial ability is poor, the infection rate after transplantation is high, and the infection not only directly causes the transplantation failure, but also threatens the life of a patient. At present, the clinically common measures for preventing infection after artificial skin transplantation mainly comprise: thoroughly cleaning wound surfaces, soaking artificial skin in disinfectant before transplantation, strengthening dressing change after transplantation, using antibiotics for the whole body and the like; although the measures can play a certain role in preventing infection after transplantation, the artificial skin cannot be endowed with antibacterial capacity, and the organ of a patient is easily damaged by using a large amount of the medicine, so that the body of the patient is negatively affected.
Disclosure of Invention
The artificial skin provided by the invention has continuous antibacterial activity, can prevent wound infection, can effectively promote wound healing of soft tissue injury, improves tissue regeneration and repair capacity, and accelerates reconstruction of dermis.
The invention provides an antibacterial artificial skin in a first aspect, which comprises a lower dermis layer, an upper dermis layer, an absorbable epidermis layer and a non-absorbable epidermis layer which are sequentially arranged from bottom to top;
the lower dermis layer is a spongy porous collagen layer;
the upper layer of the dermis is a porous acellular matrix layer; the porous acellular matrix layer at least comprises a layer of porous acellular matrix membrane adsorbed with active substances; the active substance comprises epidermal growth factor and basic fibroblast growth factor;
the absorbable epidermal layer is an antibacterial acellular matrix layer; the bacteriostatic acellular matrix layer comprises an acellular matrix membrane coated with nano mesoporous bioactive glass on the surface;
the non-absorbable skin layer is a film layer.
Preferably, the spongy porous collagen layer is obtained by freeze-drying a collagen solution;
the thickness of the spongy porous collagen layer is 0.5-5 mm; the aperture of the spongy porous collagen layer is 20-200 mu m; the porosity of the spongy porous collagen layer is greater than 90%.
Preferably, the thickness of the porous acellular matrix membrane is 0.02-0.1 mm; the porous acellular matrix membrane is provided with a plurality of through holes along the thickness direction, the diameter of each through hole is 0.1-1 mm, and the density of the through holes is 9-900/cm 2 。
Preferably, the acellular matrix membrane is 0.02-0.1 mm thick; the mass ratio of the nano mesoporous bioactive glass to the acellular matrix membrane is 1 (9-49).
Preferably, the particle size of the nano mesoporous bioactive glass is not more than 50 μm, and the pore diameter is 5-20 nm.
Preferably, the non-absorbable skin layer is a silicone rubber film or a polyvinyl alcohol film; the thickness of the non-absorbable skin layer is 0.1-0.25 mm.
In a second aspect, the present invention provides a method for preparing the antibacterial artificial skin of the first aspect, wherein the method comprises the following steps:
s1, cleaning fresh tissues, and then carrying out disinfection, degreasing and decellularization treatment to obtain a decellularized matrix membrane;
s2, freeze-drying and perforating the acellular matrix membrane, and then soaking the acellular matrix membrane in a solution containing active substances to obtain a porous acellular matrix membrane adsorbed with the active substances, namely an upper acellular matrix membrane of dermis;
s3, spreading the nano mesoporous bioactive glass on the surface of the acellular matrix membrane to obtain the acellular matrix membrane coated with the nano mesoporous bioactive glass on the surface, namely absorbing the epidermal layer acellular matrix membrane;
and S4, sequentially paving the absorbable epidermal layer acellular matrix membrane, the dermal upper layer acellular matrix membrane and a collagen solution in a mold, and after freeze drying, compounding an absorbable epidermal layer with a non-absorbable layer on the upper layer of the absorbable epidermal layer to obtain the antibacterial artificial skin.
Preferably, in step S1, the disinfection is to wash fresh tissue and then slice the tissue into slices, and soak the slices in an ethanol solution of peracetic acid for 30-60 min;
the degreasing step is that the disinfected tissues are placed in a sodium hydroxide solution to be soaked for 30-60 min and then are washed by a phosphate buffer solution;
and the cell removing treatment is to soak the degreased tissue in a trypsin solution for 1-2 h to obtain the cell-free matrix layer.
Preferably, the solution containing the active substance is obtained by mixing a collagen solution, an epidermal growth factor and a basic fibroblast growth factor;
in the solution containing the active substances, the mass fraction of collagen is 0.5-0.9%, and the concentration of the epidermal cell growth factor is 5ug/mL; the concentration of the basic fibroblast growth factor is 5ug/mL.
Preferably, the collagen solution is obtained by mixing type I collagen and an acetic acid solution; the mass fraction of acetic acid in the acetic acid solution is 4-8%.
Compared with the prior art, the invention at least has the following beneficial effects:
the upper layer of the dermis of the invention adopts a porous acellular matrix layer obtained by a tissue acellular process, has a structure similar to that of natural dermis, has stronger mechanical property, is coated with active substances, and can accelerate the reconstruction of the dermis; the lower dermis layer is a spongy porous collagen layer, which is favorable for hemostasis, fibroblast and capillary ingrowth and reconstruction of the lower dermis layer; the absorbable epidermal layer is a tissue-free cell matrix layer treated by nano mesoporous bioactive glass, can promote cell adhesion and proliferation, improve tissue regeneration and repair capability, has continuous antibacterial activity, achieves the aim of preventing wound infection, and can play a role similar to an epidermal cuticle in the later period; the non-absorbable skin layer is a silicone rubber film or a polyvinyl alcohol film, and can play a role in preventing water loss, isolating and preventing bacteria from invading.
The upper dermis layer and the lower dermis layer of the invention adopt materials with different sources and processes to achieve the control of degradation and tissue reconstruction time, and the degradation and tissue reconstruction time can be controlled through different layering layer numbers, thereby meeting different clinical requirements. The artificial skin provided by the invention has continuous antibacterial activity, can prevent wound infection, can effectively promote the healing of the wound of soft tissue injury, improves the regeneration and repair capacity of tissues, and accelerates the reconstruction of a dermis.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a schematic structural diagram of bacteriostatic artificial skin provided by the invention;
FIG. 2 is a flow chart of a preparation method of the bacteriostatic artificial skin provided by the invention;
in the figure, 1-the hypodermis, 2-the upper dermis, 3-the absorbable epidermis, 4-the non-absorbable epidermis.
Detailed Description
To make the objects, technical solutions and advantages of the embodiments of the present invention clearer and more complete, the following will clearly and completely describe the technical solutions in the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments, and all other embodiments obtained by a person of ordinary skill in the art without creative efforts based on the embodiments of the present invention belong to the protection scope of the present invention.
As shown in fig. 1, the present invention provides an antibacterial artificial skin in a first aspect, which comprises a hypodermal layer 1, an upper dermal layer 2, an absorbable epidermal layer 3 and a non-absorbable epidermal layer 4 arranged in this order from bottom to top;
the lower dermis layer 1 is a spongy porous collagen layer;
the upper dermis layer 2 is a porous acellular matrix layer; the porous acellular matrix layer at least comprises a layer of porous acellular matrix membrane adsorbed with active substances; the active substance comprises Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF);
the absorbable epidermal layer 3 is a bacteriostatic acellular matrix layer; the bacteriostatic acellular matrix layer comprises an acellular matrix membrane coated with nano mesoporous bioactive glass on the surface;
the non-absorbable skin layer 4 is a silicone rubber film or a polyvinyl alcohol film.
In the present invention, "upper" and "lower" are defined based on the positional relationship of the respective layers in fig. 1.
The upper layer of the dermis of the invention adopts a porous acellular matrix layer obtained by a tissue acellular process, has a structure similar to that of natural dermis, has stronger mechanical property, is coated with active substances, and can accelerate the reconstruction of the dermis; the lower dermis layer is a spongy porous collagen layer, which is favorable for hemostasis, fibroblast and capillary ingrowth and reconstruction of the lower dermis layer; the absorbable epidermal layer is a tissue-free cell matrix layer treated by nano mesoporous bioactive glass, can promote cell adhesion and proliferation, improve tissue regeneration and repair capability, has continuous antibacterial activity, can promote healing of wound surface of soft tissue injury while achieving the purpose of preventing wound surface infection, and can play a role similar to cuticle of epidermis in the later period; the non-absorbable skin layer is a silicon rubber film or a polyvinyl alcohol film, and can play roles in preventing water loss, isolating and preventing bacteria from invading; the artificial skin of the present invention (excluding the non-absorbable epidermal layer) has a tensile strength of not less than 5N/cm.
The upper dermis layer and the lower dermis layer of the invention adopt materials with different sources and processes to achieve the control of degradation and tissue reconstruction time, and the degradation and tissue reconstruction time can be controlled through different layering layer numbers, thereby meeting different clinical requirements, such as common skin defect, diabetic foot, bedsore and the like. Note that the non-absorbable epidermis layer was not sewn, and the absorbable epidermis layer was sewn; when the artificial skin is trimmed, a part of the non-absorbable epidermal layer can be reserved for covering, so that the part to be repaired is prevented from being directly exposed; after the vascularization of the new dermal tissue is completed, the non-absorbable epidermal layer can be removed.
According to some preferred embodiments, the spongy porous collagen layer is obtained by freeze-drying a collagen solution;
the thickness of the spongy porous collagen layer is 0.5 to 5mm (for example, it may be 0.5mm, 1mm, 1.5mm, 2mm, 2.5mm, 3mm, 3.5mm, 4mm, 4.5mm, or 5 mm); the pore diameter of the spongy porous collagen layer is 20 to 200 [ mu ] m (for example, 20 [ mu ] m, 50 [ mu ] m, 60 [ mu ] m, 80 [ mu ] m, 100 [ mu ] m, 120 [ mu ] m, 140 [ mu ] m, 160 [ mu ] m, 180 [ mu ] m, or 200 [ mu ] m); the porosity of the spongy porous collagen layer is greater than 90%.
The dermal lower layer is a spongy porous collagen layer which is of a porous structure obtained by freeze-drying a collagen solution, the aperture size of the dermal lower layer is 20-200 mu m, and the porosity is greater than 90%, so that the dermal lower layer is suitable for adhesion and migration of cells, and is beneficial to growth of connective tissues, formation of new blood vessels and skin regeneration.
According to some preferred embodiments, the porous acellular matrix membrane has a thickness of 0.02 to 0.1mm (e.g., may be 0.02mm, 0.03mm, 0.04mm, 0.05mm, 0.06mm, 0.07mm, 0.08mm, 0.09mm, or 0.1 mm); the porous acellular matrix membrane is provided with a plurality of through holes along the thickness direction, the diameter of each through hole is 0.1-1 mm (for example, the diameter can be 0.1mm, 0.2mm, 0.4mm, 0.8mm or 1 mm), and the density of the through holes is 9-900/cm 2 (for example, it may be 9/cm 2 50/cm, respectively 2 100 pieces/cm 2 150 pieces/cm 2 200 pieces/cm 2 250 pieces/cm 2 300 pieces/cm 2 350 pieces/cm 2 400 pieces/cm 2 450 pieces/cm 2 500 pieces/cm 2 550 pieces/cm 2 600 pieces/cm 2 650 pieces/cm 2 700 pieces/cm 2 750 pieces/cm 2 800 pieces/cm 2 850 pieces/cm 2 Or 900/cm 2 )。
In the invention, the density of the through holes is adjusted according to the aperture of the through holes, and the larger the aperture is, the smaller the density of the through holes is; when the diameter of the through-holes is 0.1mm, the density of the through-holes is not more than 900/cm 2 Then the method is finished; when the aperture of the through holes is 1mm, the density of the through holes is not more than 9/cm 2 That is, the adjustment can be specifically performed according to actual requirements.
The upper layer of the dermis of the invention is at least one layer of porous acellular matrix layer of a porous acellular matrix membrane adsorbed with active substances, and the porous acellular matrix membrane is obtained by punching the acellular matrix membrane; the acellular matrix membrane is obtained by an acellular process, has a structure similar to that of natural dermis, and has good mechanical property; the active substance can be applied to the healing of the wound surface of soft tissue injury, and the reconstruction of the dermis layer is accelerated; the number of layers of the porous acellular matrix membrane in the upper layer of the dermis is not particularly limited, and the porous acellular matrix membrane is designed and adjusted according to actual conditions.
According to some preferred embodiments, the acellular matrix membrane has a thickness of 0.02 to 0.1mm (e.g., may be 0.02mm, 0.03mm, 0.04mm, 0.05mm, 0.06mm, 0.07mm, 0.08mm, 0.09mm, or 0.1 mm); the mass ratio of the nano mesoporous bioactive glass to the acellular matrix membrane is 1 (9-49) (for example, the mass ratio of 1:9, 1, 15, 1, 20, 1, 25, 1.
The absorbable epidermal layer is an antibacterial acellular matrix layer of an acellular matrix membrane coated with nano mesoporous bioactive glass on the surface, and the acellular matrix membrane is obtained by an acellular process, is similar to a natural dermis structure, and has good mechanical properties; the nano mesoporous bioactive glass is prepared by adopting a sol-gel process, can promote the adhesion and proliferation of cells so as to improve the tissue regeneration and repair capacity, and can also be used as a bioactive component for promoting angiogenesis; meanwhile, the nano mesoporous bioactive glass has continuous antibacterial activity, and can promote the healing of the wound surface of soft tissue injury while achieving the purpose of preventing wound surface infection; if no nano mesoporous bioactive glass is added, the prepared artificial skin has poor bacteriostatic effect.
According to some preferred embodiments, the nano-mesoporous bioactive glass has a particle size of no greater than 50 μm and a pore size of 5 to 20nm (e.g., 5nm, 8nm, 10nm, 12nm, 15nm, 18nm, or 20 nm).
According to some preferred embodiments, the non-absorbable skin layer is a silicone rubber film or a polyvinyl alcohol film; the thickness of the non-absorbent skin layer 4 is 0.1 to 0.25mm (for example, may be 0.1mm, 0.12mm, 0.14mm, 0.16mm, 0.18mm, 0.2mm, 0.22mm, 0.24mm, or 0.25 mm).
In a second aspect, the present invention provides a method for preparing the antibacterial artificial skin according to the first aspect, as shown in fig. 2, the method comprising the following steps:
s1, cleaning fresh tissues, and then carrying out disinfection, degreasing and decellularization treatment to obtain a decellularized matrix membrane;
s2, freeze-drying and perforating the acellular matrix membrane, and then soaking the acellular matrix membrane in a solution containing active substances to obtain a porous acellular matrix membrane adsorbed with the active substances, namely an upper acellular matrix membrane of dermis;
s3, spraying the nano mesoporous bioactive glass on the surface of the acellular matrix membrane to obtain the acellular matrix membrane coated with the nano mesoporous bioactive glass, namely absorbing the epidermal layer acellular matrix membrane;
and S4, sequentially paving the absorbable epidermal layer acellular matrix membrane, the dermal upper layer acellular matrix membrane and the collagen solution in a mold, and compounding a non-absorbable epidermal layer on the upper layer of the absorbable epidermal layer after freeze drying to obtain the antibacterial artificial skin.
The freeze drying comprises a pre-freezing stage, a first sublimation stage and a second sublimation stage, wherein the target temperature and the target time length of each stage are as follows:
a pre-freezing stage: freezing at-30 deg.C for 30min without adding sample, and then freezing at-30 deg.C for 90min;
a first sublimation stage: vacuumizing, aerating at 100 + -10 Pa, freezing at-10 deg.C for 60min, and freezing at 0 deg.C for 120min;
a second sublimation stage: vacuumizing, aerating at 100 + -10 Pa, freezing at 10 deg.C for 120min, freezing at 20 deg.C for 120min, and freezing at 25 deg.C for 120min; and (5) turning off the machine when the temperature of the front incubator reaches 25 ℃.
The collagen solution is prepared by adopting fresh tissues (small intestines of pigs, amnion, dura mater or bladder tissues) to carry out a decellularization process to obtain a decellularized matrix membrane, perforating the surface of the obtained decellularized matrix membrane by using a perforating tool after freeze-drying, and then soaking the decellularized matrix membrane in a solution containing active substances to obtain a porous decellularized matrix membrane adsorbed with the active substances, namely a dermal upper layer decellularized matrix membrane; then, the surface of the acellular matrix membrane prepared by adopting fresh tissues (small intestines of pigs, amnion, dura mater or bladder tissues) to carry out an acellular process is coated with nano mesoporous bioactive glass to obtain the acellular matrix membrane coated with the nano mesoporous bioactive glass, namely the acellular matrix membrane of the epidermis layer can be absorbed; and finally, sequentially paving the absorbable epidermal layer acellular matrix membrane, the dermal upper layer acellular matrix membrane and the collagen solution in a mould, and after integral freeze drying, compounding the non-absorbable epidermal layer on the absorbable epidermal layer by adopting a medical adhesive coating to obtain the antibacterial artificial skin, wherein the medical adhesive coating is a silica gel adhesive layer, an acrylic acid coating or a polyurethane coating.
The invention can adjust the number of different layers of the upper layer of the dermis in the layering process, control the time of degradation and tissue reconstruction and meet different clinical requirements.
According to some preferred embodiments, in step S1, the disinfection is to wash fresh tissue and then slice the tissue into ethanol solution of peracetic acid for 30-60 min (for example, 30min, 35min, 40min, 45min, 50min, 55min or 60min may be allowed); in the ethanol solution of peracetic acid of the present invention, the mass fraction of peracetic acid is 0.2%.
The degreasing step is to soak the disinfected tissue in a sodium hydroxide solution for 30-60 min (for example, 30min, 35min, 40min, 45min, 50min, 55min or 60 min), and then to clean the tissue with a phosphate buffer; the sodium hydroxide solution of the present invention was an aqueous sodium hydroxide solution having a concentration of 15mmol/L.
The decellularization treatment is to soak the degreased tissue in a trypsin solution for 1 to 2 hours (for example, 1 hour, 1.2 hours, 1.4 hours, 1.6 hours, 1.8 hours or 2 hours) to obtain a decellularized matrix layer; the trypsin solution of the present invention may have a mass fraction of 0.25 to 0.5% (for example, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5%).
According to some preferred embodiments, the active substance-containing solution is obtained by mixing a collagen solution, epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF);
in the solution containing the active substances, the mass fraction of collagen is 0.5-0.9%, and the concentration of the Epidermal Growth Factor (EGF) is 5ug/mL; the concentration of the basic fibroblast growth factor (bFGF) is 5ug/mL.
According to some preferred embodiments, the collagen solution is obtained by mixing type I collagen and an acetic acid solution; the mass fraction of acetic acid in the acetic acid solution is 4 to 8% (for example, may be 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, or 8%).
In the present invention, the preparation method of type I collagen is referred to patent CN114732954a, which includes: s1: removing fat and fascia from bovine Achilles tendon, cleaning, and freezing; s2: cutting the frozen bovine achilles tendon to a thickness of about 0.8 mm; s3: soaking the slices cut by the S2 in a sodium bicarbonate solution with the mass fraction of 1.0-1.5% for 12-24 h, and then washing the slices with purified water for a plurality of times to degrease; s4: adding the degreased slices of S3 into an acid solution with pH of 1-3, carrying out enzymolysis for 48-96 h at the temperature of 0-25 ℃, and removing terminal peptides to obtain an enzymolysis solution; the acid solution is prepared from one or more of acetic acid, citric acid and phosphoric acid; in the invention, the acid solution with pH of 2 is preferably used for enzymolysis, because the optimal conditions for activating the added pepsin are normal temperature, pH value is 2-3, and the normal temperature enzymolysis is 48-96 h, preferably 96h, the enzymolysis is complete and the yield is high; s5: centrifuging the enzymolysis liquid obtained in the step S4 by using a centrifuge, taking supernatant, and separating out collagen floccule by using a salt solution, wherein the salt is one or more of sodium chloride, potassium chloride, sodium carbonate and potassium carbonate, and the salt solution is, for example, a supersaturated salt solution; s6: adding the white floccule obtained in the step S5 into a dialysis bag for dialysis, and in order to ensure that the environmental change of the external dialysis liquid is mild and avoid the change of the external dialysis liquid from being too severe so as to cause irreversible precipitation of collagen in the internal dialysis liquid, adopting a gradient dialysis mode, such as gradually reducing the concentration of acetic acid in the external liquid, and finally using purified water for dialysis to complete the purification of the collagen; the way of gradient dialysis may be, for example: firstly, putting a dialysis bag into 45L of dialysis solution with pH =3 for dialysis for 4 days, wherein the dialysis temperature is 15 +/-3 ℃, and the dialysis solution is replaced for 1 time every 2 days; then placing the dialysis bag in 45L of dialysate with pH =4, dialyzing for 5 days at 15 + -3 deg.C, and replacing the dialysate every 1 day for 1 time; dialyzing the dialysis bag in 45L purified water at 15 + -3 deg.C for 6 days, and changing the dialysate 3 times per day, once in the morning, at noon and at night; in the present invention, the pH =3 dialysate and the pH =4 dialysate are both, for example, acetic acid solutions prepared from acetic acid and purified water, and the pH =3 dialysate may be prepared, for example, by: 45000mL of purified water is added into a dialysis tank, 146.694mL of acetic acid is accurately taken by a measuring cylinder and a pipette and added into the dialysis tank, and the mixture is uniformly stirred to obtain an acetic acid solution with pH = 3; the dialysate with pH =4 can be formulated, for example, as: 45000mL of purified water is added into a dialysis tank, 1.479mL of acetic acid is accurately taken by a liquid transfer gun and added into the dialysis tank, and the mixture is uniformly stirred, so that an acetic acid solution with the pH =4 is obtained; s7: placing the collagen gel with certain mass and solid content produced in the step S6 into a homogenizer, adding water with certain mass to prepare a collagen solution with the concentration of 0.5-0.9%, wherein the homogenizing frequency is 20-50 Hz, and the homogenizing time is 15-60min; s8: and (4) freeze-drying the homogenized collagen gel obtained in the step (S7) to obtain the I-type collagen with uniform texture.
In order to more clearly illustrate the technical solutions and advantages of the present invention, the present invention is further described below with reference to the following embodiments.
The materials and reagents in the invention can be obtained by direct purchase or self-synthesis on the market, and the specific model is not limited.
The following methods were used for the performance tests of the examples and comparative examples of the present invention:
bacteriostatic and microbial barrier test
1) Equipment
(1) Test microorganism, staphylococcus aureus (ATCC 6538)
(2) Culture medium including blood agar, nutrient agar, and glucose nutrient broth
(3) Artificial skin: area 50mm x 50mm
2) Procedure for the preparation of the
(1) The golden yellow staphylococcus is inoculated in 6mL of glucose nutrient broth culture medium, and the viable bacteria count is carried out on the bacterial suspension which is cultured for 16h at 37 ℃.
(2) The artificial skin was spread in a sterile dish with the epidermis facing up.
(3) By using a container 10 7 The cfu/mL staphylococcus aureus suspension is dripped on the artificial skin without contacting with 10 drops of 0.01mL each.
(4) The infected artificial skin is placed under the conditions of temperature of 20-25 ℃ and relative humidity of 40-50% to be dried for no more than 6h.
(5) And (3) flatly paving the infected artificial skin on the surface of the blood agar culture medium, completely contacting, enabling the infected surface to be upward, and removing the artificial skin after 5-6 s.
(6) The blood agar culture medium is cultured at 37 ℃ for 16-24 h for colony counting.
3) And (3) reporting a result: the number of colonies grown on each blood agar medium plate and the total number of colonies grown on 3 plates.
The release performance test method comprises the following steps: a test sample (artificial skin of example or comparative example) with the size of 20X 20 (mm) is taken in 10mL of simulated body fluid containing collagenase I0.04 Unit/mL, placed at 37 ℃, and periodically sampled to measure the contents of EGF and bFGF in the simulated body fluid by an ELISA method, so as to determine the release amount of Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF) along with the change of time.
The mechanical property testing method comprises the following steps: the test samples (the portions of the artificial skin of the examples and comparative examples except for the non-absorbable layer) were taken, prepared into rectangular test specimens of 1cm × 3cm, having a tensile strain rate of 10mm/min, and their maximum tensile strength was measured on a mechanical tester until the product was broken.
In vitro cell complex culture
1. In vitro cell planting
(1) Cutting the artificial skin into a disc shape with the diameter of 6mm by using a puncher;
(2) Putting the prepared disc-shaped material into another culture dish, adding a proper amount of DMEM culture solution containing 10% fetal calf serum to completely submerge the disc-shaped material, and incubating for 24 hours in a constant-temperature incubator at 37 ℃;
(3) Mice fibroblasts, NIH/3T3, were digested with 0.25% trypsin and then counted on a cell counting plate. Centrifuging at 500rpm for 5min after cell digestion, discarding supernatant, adding 5mL DMEM culture solution containing 10% fetal calf serum, centrifuging, resuspending, diluting cells, and adjusting concentration to 8 × 10 4 /mL。
(4) Taking the prepared materials out of the incubator, placing the materials in a super clean bench, clamping and transferring the materials to a 96-well plate by using sterile forceps, and placing the materials at the bottom;
(5) Add 100. Mu.L of cell suspension to each well, inoculate 96-well plates in groups of 4 duplicate wells (one for microscopic observation and the other three for cell viability detection) based on experimental material, add only control wells containing no material in cell culture medium and blank wells containing no cells in medium, and place in a cell incubator overnight for culture.
(6) At 37 ℃,5% CO 2 Incubating in a constant-temperature incubator, and changing the liquid every 2 to 3 days.
CCK-8 Absorbance assay
(1) To eliminate the effect of cells not adhering to the scaffold, the sample was transferred to a new 96-well plate.
(2) Selecting 20mL of a centrifuge tube, adding 4500uL of DMEM culture solution and 500uL of CCK-8 solution, uniformly stirring, and taking 100uL of mixed solution, wherein the DMEM culture medium is 90uL of the solution 10uL of CCK-8 was added to each well, mixed well, and then subjected to 5% CO 2 Incubate at 37 ℃ for 2h.
(3) The treated culture medium is sucked out and transferred into another 96-well plate, each well has 100uL, and the absorbance at 450nm is detected by an enzyme-labeling instrument.
3. Microscopic observation and detection of fibroblast and artificial skin culture
(1) After 5 days of incubation, the medium was aspirated and washed three times with PBS (10 mm, ph = 7.4).
(2) The epidermal layer and the dermal upper layer were separated from the artificial skin, and cell adhesion was observed on both sides of the dermal upper layer and on the lower side of the epidermal lower layer (absorbable epidermal layer).
Example 1
An antibacterial artificial skin comprises a lower dermis layer 1, an upper dermis layer 2, an absorbable epidermal layer 3 and a non-absorbable epidermal layer 4 which are sequentially arranged from bottom to top; the lower dermis layer 1 is a spongy porous collagen layer with the thickness of 2.5mm, the aperture of 100 mu m and the porosity of 93 percent; the upper dermis layer 2 is three layers of porous acellular matrix membranes adsorbing active substances, the thickness of a single-layer porous acellular matrix membrane is 0.1mm, a plurality of through holes are arranged in the porous acellular matrix membrane along the thickness direction, the aperture of each through hole is 0.1mm, and the density of each through hole is 500/cm 2 The active substance comprises Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF); the absorbable epidermal layer 3 is an acellular matrix membrane of which the surface is coated with nano-mesoporous bioactive glass, the thickness of the single-layer acellular matrix membrane is 0.05mm, the mass ratio of the nano-mesoporous bioactive glass to the acellular matrix membrane is 1:9, the particle size of the nano-mesoporous bioactive glass is 40 mu m, and the pore diameter is 10nm; the non-absorbable skin layer 4 was a silicone rubber film having a thickness of 0.15 mm.
Example 2
An antibacterial artificial skin comprises a lower dermis layer 1, an upper dermis layer 2, an absorbable epidermis layer 3 and a non-absorbable epidermis layer 4 which are sequentially arranged from bottom to top; the dermal lower layer 1 is a spongy porous collagen layer with the thickness of 2.5mm, the aperture of 100 mu m and the porosity of 93 percent; the upper layer 2 of dermis is three layers of porous acellular matrix membranes adsorbed with active substances, and the upper layer of dermis is a single layer of porous acellular membranesThe thickness of the matrix membrane is 0.1mm, the porous acellular matrix membrane is provided with a plurality of through holes along the thickness direction, the aperture of the through holes is 0.1mm, and the density is 500/cm 2 The active substance comprises Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF); the absorbable epidermal layer 3 is an acellular matrix membrane of which the surface is coated with nano-mesoporous bioactive glass, the thickness of the single-layer acellular matrix membrane is 0.05mm, the mass ratio of the nano-mesoporous bioactive glass to the acellular matrix membrane is 1; the non-absorbable skin layer 4 was a silicone rubber film having a thickness of 0.15 mm.
Example 3
An antibacterial artificial skin comprises a lower dermis layer 1, an upper dermis layer 2, an absorbable epidermis layer 3 and a non-absorbable epidermis layer 4 which are sequentially arranged from bottom to top; the lower dermis layer 1 is spongy porous collagen layer with the thickness of 2.5mm, the aperture of 100 mu m and the porosity of 93 percent; the upper layer 2 of dermis is three layers of porous acellular matrix membranes adsorbed with active substances, the thickness of a single layer of porous acellular matrix membrane is 0.1mm, a plurality of through holes are arranged in the porous acellular matrix membrane along the thickness direction, the aperture of each through hole is 0.1mm, and the density of each through hole is 500/cm 2 The active substance comprises Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF); the absorbable epidermal layer 3 is an acellular matrix membrane of which the surface is coated with nano-mesoporous bioactive glass, the thickness of the single-layer acellular matrix membrane is 0.05mm, the mass ratio of the nano-mesoporous bioactive glass to the acellular matrix membrane is 1; the non-absorbent skin layer 4 was a polyvinyl alcohol film having a thickness of 0.15 mm.
Example 4
An antibacterial artificial skin comprises a lower dermis layer 1, an upper dermis layer 2, an absorbable epidermis layer 3 and a non-absorbable epidermis layer 4 which are sequentially arranged from bottom to top; the lower dermis layer 1 is a spongy porous collagen layer with the thickness of 0.5mm, the pore diameter of 20 mu m and the porosity of 91 percent; the upper layer 2 of dermis is a layer of porous acellular matrix membrane adsorbed with active substances, the thickness of the single-layer porous acellular matrix membrane is 0.02mm, and the porous acellular matrix layer is in the thickness directionA plurality of through holes are arranged in the groove, the aperture of each through hole is 1mm, and the density is 9/cm 2 (ii) a The active substance comprises Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF); the absorbable epidermal layer 3 is a acellular matrix membrane coated with nano-mesoporous bioactive glass on the surface, the thickness of the single-layer acellular matrix membrane is 0.02mm, the mass ratio of the nano-mesoporous bioactive glass to the acellular matrix membrane is 1:9, the particle size of the nano-mesoporous bioactive glass is 40 mu m, and the pore diameter is 5nm; the non-absorbable skin layer 4 was a polyvinyl alcohol film having a thickness of 0.1mm.
Example 5
An antibacterial artificial skin comprises a lower dermis layer 1, an upper dermis layer 2, an absorbable epidermis layer 3 and a non-absorbable epidermis layer 4 which are sequentially arranged from bottom to top; the lower dermis layer 1 is a spongy porous collagen layer with the thickness of 5mm, the pore diameter of 200 mu m and the porosity of 93 percent; the upper dermis layer 2 is two layers of porous acellular matrix membranes adsorbed with active substances, the thickness of a single-layer porous acellular matrix membrane is 0.1mm, the porous acellular matrix layer is provided with a plurality of through holes along the thickness direction, the aperture of the through holes is 0.5mm, and the density is 200/cm 2 (ii) a The active substance comprises Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF); the absorbable epidermal layer is a acellular matrix membrane of which the surface is coated with nano-mesoporous bioactive glass, the thickness of the single-layer acellular matrix membrane is 0.1mm, the mass ratio of the nano-mesoporous bioactive glass to the acellular matrix membrane is 1:9, the particle size of the nano-mesoporous bioactive glass is 40 mu m, and the aperture is 20nm; the non-absorbable skin layer 4 was a silicone rubber film having a thickness of 0.25mm.
Comparative example 1
Comparative example 1 is essentially the same as example 1 except that: the absorbable epidermal layer is an acellular matrix film, and the thickness of the acellular matrix film is 0.1mm.
The surface of the absorbable epidermis layer is not coated with the nano mesoporous bioactive glass, so that the bacteriostatic and cell adhesion promoting capabilities are weakened.
Comparative example 2
Comparative example 2 is essentially the same as example 1, except that: the mass ratio of the nano mesoporous bioactive glass to the acellular matrix membrane in the absorbable epidermal layer is 1.
The dosage of the nano mesoporous bioactive glass coated on the surface of the absorbable epidermis layer is less, and the bacteriostatic and cell adhesion promoting capabilities are weakened.
Comparative example 3
Comparative example 3 is essentially the same as example 1 except that: the mass ratio of the nano mesoporous bioactive glass to the acellular matrix membrane in the absorbable epidermal layer is 1:5.
The nano mesoporous bioactive glass has too much consumption and is not easy to be completely coated on the surface of the acellular matrix membrane.
Comparative example 4
Comparative example 4 is substantially the same as example 1 except that: the upper layer of the dermis is three layers of porous acellular matrix membranes, the thickness of the single layer of porous acellular matrix membrane is 0.1mm, a plurality of through holes are arranged in the porous acellular matrix membrane along the thickness direction, the aperture of each through hole is 0.1mm, and the density of each through hole is 500/cm 2 。
Active substances are not adsorbed on the surface of the porous acellular matrix membrane, so that the cell proliferation capacity is weakened, and the tissue repair and reconstruction are not facilitated.
Comparative example 5
Comparative example 5 is substantially the same as example 1 except that: the upper layer of the dermis is three acellular matrix membranes adsorbed with active substances, and the thickness of a single acellular matrix membrane is 0.1mm.
Because the acellular matrix membrane is not perforated, cells cannot pass through the upper layer of the dermis, and the tissue repair and reconstruction time is prolonged.
Comparative example 6
Comparative example 6 is substantially the same as example 1 except that: the hypodermis is a spongy porous collagen layer with porosity of 75%.
The porosity of the dermis is too small, the cell proliferation capacity is weakened, and the repair and reconstruction of the dermis are influenced.
Comparative example 7
Comparative example 7 is substantially the same as example 1 except that: there is no unabsorbable epidermal layer.
The microbial barrier capability of the non-absorbable epidermal layer is reduced, and the infection risk is increased.
TABLE 1 bacteriostatic and microbial barrier test data for artificial skin prepared in the inventive examples and comparative examples.
As can be seen from table 1, the absorbable surface layers of examples 1 to 5 and comparative examples 3 to 6 of the present invention are coated with the nano mesoporous bioactive glass in the dosage range of the present invention and are provided with the non-absorbable surface layer, and the surface layer in the artificial skin has excellent antibacterial and microbial barrier properties; comparative example 1 can absorb the surface of the epidermis and not spread the biological active glass of nanometer mesopore, the bacteriostatic effect is bad; comparative example 2 the surface of the absorbable skin layer is coated with a small amount of nano mesoporous bioactive glass and is provided with a non-absorbable layer, so that the absorbable skin layer has a certain bacteriostatic action; in comparative example 7, the antibacterial effect is poor because no non-absorbable surface layer is arranged; therefore, the artificial skin can be ensured to have excellent antibacterial and microbial barrier properties only by spreading the nano mesoporous bioactive glass on the surface of the absorbable skin layer and arranging the non-absorbable surface layer.
Table 2. Release process data of Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF) in artificial skin (excluding non-absorbable epidermal layer) prepared in the examples of the present invention and comparative example.
As can be seen from Table 2, the release of Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF) in the upper layer of dermis of the bacteriostatic artificial skin is fast in the first few days, and becomes slow in the later days, so that the release of the EGF and the bFGF can be well matched with natural repair of wounds, and the skin repair process can be promoted.
TABLE 3 mechanical test data for artificial skin prepared in the examples of the present invention and comparative examples (excluding the non-absorbable epidermal layer).
As can be seen from Table 3, the bacteriostatic artificial skin disclosed by the invention has good mechanical properties.
TABLE 4. Experimental data of in vitro cell complex culture of artificial skin prepared in the examples of the present invention and comparative examples.
It should be noted that "++++" indicates a large amount of cell growth, "+++" indicates a small amount of cell growth, "+" indicates a very small amount of cell growth, "-" no cell growth: the inner surface of the absorbable epidermal layer is one surface close to the upper dermis layer, and the outer surface of the upper dermis layer is one surface close to the absorbable epidermal layer; the inner surface of the dermis is close to the lower surface of the dermis; the magnitude of the OD value reflects the strength of the cell proliferation promoting capacity, and the larger the OD value is, the stronger the cell proliferation capacity is.
As can be seen from the comparison between examples 1-5 and comparative examples, in comparative examples 1-2, the surface of the resorbable epidermis layer is not coated or is coated with little or no nano-mesoporous bioactive glass, and only a very small amount of cells grow on the lower surface of the resorbable epidermis layer; comparative example 4 the surface of the porous acellular matrix membrane in the upper dermis layer did not adsorb active substances, the cell proliferation ability was reduced, and only a very small amount of cells grew on both the lower surface of the absorbable epidermis layer and the upper surface of the upper dermis layer; comparative example 5 the acellular matrix membrane in the upper dermis layer was not perforated, cells could not pass through the upper dermis layer, and the lower surface of the absorbable epidermis layer grew without cells; in comparative example 6, the porosity of the lower dermal layer was too small, the cell proliferation ability was weakened, the upper surface of the upper dermal layer had very little cell growth, and the lower surface of the absorbable epidermal layer had very little cell growth.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. The antibacterial artificial skin is characterized by comprising a lower dermis layer, an upper dermis layer, an absorbable epidermis layer and a non-absorbable epidermis layer which are sequentially arranged from bottom to top;
the lower dermis layer is a spongy porous collagen layer;
the upper layer of the dermis is a porous acellular matrix layer; the porous acellular matrix layer at least comprises a layer of porous acellular matrix membrane adsorbed with active substances; the active substance comprises epidermal growth factor and basic fibroblast growth factor;
the absorbable epidermal layer is a bacteriostatic acellular matrix layer; the bacteriostatic acellular matrix layer comprises an acellular matrix membrane coated with nano mesoporous bioactive glass on the surface;
the non-absorbable skin layer is a film layer.
2. The antibacterial artificial skin according to claim 1, wherein the spongy porous collagen layer is obtained by freeze-drying a collagen solution;
the thickness of the spongy porous collagen layer is 0.5-5 mm;
the aperture of the spongy porous collagen layer is 20-200 mu m; the porosity of the spongy porous collagen layer is greater than 90%.
3. The antibacterial artificial skin according to claim 1, wherein the thickness of the porous acellular matrix membrane is 0.02 to 0.1mm;
the porous acellular matrix membrane is provided with a plurality of through holes along the thickness direction, the diameter of each through hole is 0.1-1 mm, and the density of the through holes is 9-900/cm 2 。
4. The antibacterial artificial skin according to claim 1, wherein the thickness of the acellular matrix membrane is 0.02 to 0.1mm;
the mass ratio of the nano mesoporous bioactive glass to the acellular matrix membrane is 1 (9-49).
5. The antibacterial artificial skin according to claim 1, wherein the nano mesoporous bioactive glass has a particle size of not more than 50 μm and a pore size of 5-20 nm.
6. The antimicrobial artificial skin of claim 1, wherein the non-absorbable skin layer is a silicone rubber film or a polyvinyl alcohol film; the thickness of the non-absorbable skin layer is 0.1-0.25 mm.
7. A method of preparing an antimicrobial artificial skin according to any one of claims 1 to 6, comprising the steps of:
s1, cleaning fresh tissues, and then carrying out disinfection, degreasing and decellularization treatment to obtain a decellularized matrix membrane;
s2, freeze-drying and perforating the acellular matrix membrane, and then soaking the acellular matrix membrane in a solution containing active substances to obtain a porous acellular matrix membrane adsorbed with the active substances, namely an upper acellular matrix membrane of dermis;
s3, spraying the nano mesoporous bioactive glass on the surface of the acellular matrix membrane to obtain the acellular matrix membrane coated with the nano mesoporous bioactive glass, namely absorbing the epidermal layer acellular matrix membrane;
and S4, sequentially paving the absorbable epidermal layer acellular matrix membrane, the dermal upper layer acellular matrix membrane and the collagen solution in a mold, and compounding a non-absorbable epidermal layer on the upper layer of the absorbable epidermal layer after freeze drying to obtain the antibacterial artificial skin.
8. The preparation method according to claim 7, wherein in step S1, the disinfection is that the fresh tissue is washed and sliced and soaked in an ethanol solution of peroxyacetic acid for 30-60 min;
the degreasing is to put the sterilized tissue into a sodium hydroxide solution to be soaked for 30-60 min and then to be washed by a phosphate buffer solution;
and the cell removing treatment is to soak the degreased tissue in a trypsin solution for 1-2 h to obtain the cell-free matrix layer.
9. A method for preparing a composition according to claim 7, wherein the solution containing the active substance is obtained by mixing a collagen solution, an epidermal growth factor and a basic fibroblast growth factor;
in the solution containing the active substances, the mass fraction of collagen is 0.5-0.9%, and the concentration of the epidermal cell growth factor is 5ug/mL; the concentration of the basic fibroblast growth factor is 5ug/mL.
10. A method according to claim 9, wherein the collagen solution is obtained by mixing type I collagen with an acetic acid solution; the mass fraction of acetic acid in the acetic acid solution is 4-8%.
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