CN116688234A - Preparation of active biological material of composite stem cells and application of active biological material in artificial skin - Google Patents
Preparation of active biological material of composite stem cells and application of active biological material in artificial skin Download PDFInfo
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- CN116688234A CN116688234A CN202310055382.7A CN202310055382A CN116688234A CN 116688234 A CN116688234 A CN 116688234A CN 202310055382 A CN202310055382 A CN 202310055382A CN 116688234 A CN116688234 A CN 116688234A
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- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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Abstract
The invention relates to a preparation method of an active biological material of a composite stem cell and application thereof in artificial skin, which effectively solves the problems of higher preparation cost, complex preparation process and no application in active substance research of the existing method.
Description
Technical Field
The invention relates to the technical field of bionic high polymer materials, in particular to preparation of an active biological material of a composite stem cell and application of the active biological material in artificial skin.
Background
Understanding: the skin is the organ with the largest area of human body, and has important protection function and physiological function. The serious damage to skin caused by burns, wounds, refractory skin ulcers (such as diabetic foot) and the like is a serious medical and public health problem commonly faced by all countries in the world today. In clinical practice, natural skin or artificial skin is transplanted on the surface of the wound surface which is difficult to heal and large-area wound repair.
Natural skin (including autologous skin, allogenic skin and xenogenic skin) has a structure and function close to that of recipient skin, but has problems of insufficient skin source, immune rejection, virus transmission and the like, so that the application is limited in large-area wound repair.
Artificial skin refers to a skin substitute that has been artificially developed in vitro using engineering and cellular biology principles and methods to repair and replace defective skin tissue. The artificial skin is similar to human skin in height, has the special effect of relieving pain of patients in the aspect of treating burns and scalds, does not leave scars after healing, and has good curative effect on treating chronic refractory ulcers such as diabetic feet and the like. The internationally commercialized artificial skin with dermis substitution function adopts animal-derived biological materials similar to human skin, but due to the xenogeneic nature of the biological polymers, a certain degree of immune rejection (such as inflammatory reaction) can be induced after transplantation, the rapid healing of wound surfaces is affected, and the risk of systemic inflammatory reaction syndrome is induced during large-area transplantation.
An artificial skin in a true sense should have a three-dimensional structure of the native skin and realize the function of natural skin tissue. In addition, it should also support angiogenesis and provide support for cells that are present in the local environment. It must also be able to bind to the host with minimal scarring after it is implanted in the body, while producing a controlled inflammatory response. Clinically, a large-area full-thickness skin defect is a troublesome problem in global wound medicine, and once sudden wounds, car accidents and other accidents occur, the life health and the life quality of human beings are greatly influenced. The development of stem cells and tissue engineering brings revolutionary transformation to the treatment of tissue injury and organ failure in humans, leading to a new round of medical revolution after drugs and surgery. Autologous skin grafting has been widely used at present as an important means for skin treatment of full-thickness skin defects, the earliest product representative being skin soothing. However, there is a risk of infection due to delayed wound healing, and little generation of skin appendages, due to insufficient vascularization or failure of skin grafting. In addition, the interruption of blood transport caused by microbial-infected wounds and vascular defects is one of the major factors leading to complications of full-thickness skin defects. Currently, no single skin substitute has been demonstrated on the market to fully restore normal skin structure and physiological function. Is not in place compared with the current market products.
Various types of artificial skin product brands that have been marketed today include Dermagraft, integra, alloDerm, matriStem, priMatrix, PELNAC and PermeaDerm skin dressings; and EpiSkin, epiDerm, matriDerm, and tissue engineering composite skin (artificial skin) such as Apligraf and skin care (actigspin).
And (5) searching: the related field is searched in a patent library, and tens of related patents are searched, wherein the technical problems which are similar to the technical problems solved by the application are as follows.
Citation:
for the application patent with the publication number of CN110960730B, a bionic anti-rejection artificial skin for 3D printing and a preparation method thereof are disclosed. The method comprises the steps of dissolving polysaccharide substances with amino groups and proteins in an acidic solution to obtain a precursor solution, dissolving spermidine and small molecular dialdehyde or a high molecular material with two ends modified by aldehyde groups in absolute ethyl alcohol to obtain a spermidine cross-linking agent, printing the precursor solution in a culture dish by adopting a 3D biological printing method, atomizing the spermidine cross-linking agent, sequentially performing, controlling the hardness of each layer by controlling the concentration of the spermidine cross-linking agent of each layer, and finally obtaining the bionic rejection-resistant artificial skin with gradient change of hardness.
For the invention patent with the publication number of CN111228572B, an artificial skin and a preparation method and application thereof are disclosed. The method comprises the following steps: uniformly coating liquid silicone rubber on the surface of a solid support, and performing first curing treatment to form an uncured silicone rubber layer; directly placing the formed collagen film on the surface of the non-cured silicone rubber layer in the step S1, and performing second curing treatment to completely cure the silicone rubber layer and tightly bond the silicone rubber layer with the collagen film; separating the fully cured silicone rubber layer bonded to the collagen membrane in the step from the solid support to obtain the artificial skin.
For the invention patent with the publication number of CN111407929A, a novel artificial skin and a preparation method thereof are provided. The preparation method of the novel artificial skin specifically comprises the following steps: taking fresh animal isolated skin, obtaining acellular dermal matrix through a series of treatments of sodium hydroxide, DNase, trypsin, sodium dodecyl sulfate, peracetic acid, PBS and the like, uniformly mixing graphene oxide with acellular dermal matrix solution through ultrasonic mixing, and obtaining the novel artificial skin through enrichment treatment.
For the invention patent with the publication number of CN111494713A, a micro-nano bionic artificial skin repair membrane is disclosed, which comprises a basal membrane, a bracket layer, a surface membrane and a separable surface membrane; the substrate film, the bracket layer, the polyurethane surface film and the separable surface film are sequentially laminated from inside to outside; the substrate film is a non-woven film formed by electrostatic spinning and spraying of sericin; the diameter of the sericin electrostatic spinning is 50-500nm; the support layer is a non-woven layer formed by blending electrostatic spinning and spraying; the blended electrostatic spinning is a spiral fiber yarn formed by blending PLA and PCL; the diameter of the blended electrostatic spinning is 0.2-5 mu m; the epidermis film is a biodegradable polyurethane film; the separable surface film is woven by polypropylene fiber.
For the invention patent with the publication number of CN113174138A, an artificial skin membrane containing lamellar liquid crystal, a preparation method and application thereof are disclosed. The artificial skin membrane comprises a base material and a lyotropic liquid crystal component, wherein the base material is a network structure formed by polydimethylsiloxane and chitosan, and the mass percentage of the lyotropic liquid crystal component in the artificial skin membrane is 3-8%. The lyotropic liquid crystal component is one or more selected from glycoside liquid crystal components, sucrose ester liquid crystal components, lecithin liquid crystal components, phosphate ester liquid crystal components, stearoyl liquid crystal components and fatty acid ester liquid crystal components.
For the invention patent with the publication number of CN113105711A, an artificial skin simulating perspiration is provided, and comprises a water permeable layer and a temperature sensitive polyurethane hydrogel layer. The preparation method comprises the following steps: weighing the carbon nano tube, polyether polyol, isocyanate, a chain extender, N-ethylmorpholine, tantalum pentoxide, pentaerythritol, stannous octoate and disodium ethylene diamine tetraacetate magnesium salt according to parts by weight; emulsifying to obtain a carbon nano tube emulsion; ozone gas is introduced to obtain hydrophilic carbon nanotubes; placing the modified carbon nano tube, polyether polyol, isocyanate, N-ethylmorpholine and tantalum pentoxide into a reaction kettle, and setting reaction conditions to prepare a polyurethane semi-finished product; adding a chain extender, pentaerythritol and stannous octoate into a reaction kettle, and adding the chain extender, the pentaerythritol and the stannous octoate into the reaction kettle to prepare a polyurethane prepolymer; the polyurethane prepolymer and disodium ethylene diamine tetraacetate magnesium salt further obtain the temperature-sensitive polyurethane hydrogel.
For the invention patent with the publication number of CN113577398A, a 3D printing artificial skin and a preparation method thereof are disclosed, wherein the skin comprises the following components: an epidermis layer and a dermis layer; the epidermis layer is covered on the upper surface of the dermis layer, is in a hydrogel state and has pH sensitivity; the components of the skin layer include: the first gelatin, tannic acid and ferric salt improve the mechanical property of the epidermis and have long-acting stable antibacterial and anti-inflammatory properties; solves the problem that the antibacterial substances of the epidermis layer are burst released to cause cytotoxicity to normal cells in the prior art.
For the invention patent with the publication number of CN114108177A, an artificial skin material capable of releasing the growth factors in a stepwise manner by photo-thermal triggering, a preparation method and application thereof are disclosed. Relates to an artificial skin material capable of releasing growth factors in a stepwise manner by photo-thermal triggering and a preparation method thereof. The method comprises the steps of taking a degradable polymer material, a degradable natural polymer and a phase change material as main raw materials, adding antibacterial drugs and various growth factors, preparing a scaffold material with a multistage repairing effect through electrostatic spinning, and adding different antibacterial drugs into the material to realize multiple antibacterial effects; phase change material particles containing different kinds of growth factors are deposited between two fiber layers, so that the space-time controllable ordered release of the various growth factors can be triggered by light and heat, and microenvironments required by different stages of wound healing are provided.
For the patent of the invention with the publication number of CN 113797388A, an artificial skin membrane of chitin and a preparation method thereof are disclosed, chitin powder is taken as a raw material by utilizing a low-temperature freeze thawing method, the influence of sodium hydroxide concentration, chitin consumption, urea concentration and the like on the formation of a membrane material is researched, the influence of composite material types, proportions and the like on the mechanical properties of the composite material membrane is researched, and the preparation conditions are optimized to prepare the multifunctional membrane material with proper strength, degradation rate and mechanical properties.
The invention patent with publication number of CN114288474A discloses a dermis layer for promoting hair follicle regeneration, artificial skin and a preparation method thereof. The raw materials of the dermis layer comprise: collagen, YAP protein inhibitors and growth factor microspheres effectively block the activation of jagged genes in wounds by using YAP protein inhibitors, thus making the restoration of secondary skin components (hair follicles and glands), extracellular matrix structure and tensile strength indistinguishable from intact skin.
For the invention patent with publication number CN114470338A, a dermis layer, an artificial skin and a preparation method thereof are disclosed. The raw materials of the dermis layer comprise: the recombinant human collagen, the verteporfin and the enzyme have the advantages that the verteporfin in raw materials provides an effective mechanical transduction mechanism, the expression of En1 in the wound healing process is regulated, the restoration of the normal dermis ultrastructure is induced, and the problems that scars are generated in the skin healing process and hair follicles, sebaceous glands and other dermis appendages cannot be generated are solved.
For the invention patent with the bulletin number of CN214881593U, a multifunctional artificial skin culture medical instrument is disclosed, which comprises a device main body; the skin piece to be cultivated is placed on the nylon net, then the first rotating hand is rotated, the unidirectional screw rod is driven to rotate through the first rotating hand, the unidirectional screw rod rotates to enable the first sliding block to move downwards in the first sliding groove, so that the position of the nylon net can be lowered, the skin piece placed on the nylon net can be completely immersed into the culture solution, the skin piece floats in the culture solution to be cultivated, after a period of cultivation, the first rotating hand is reversely rotated, the unidirectional screw rod is reversely rotated, so that the first sliding block can move upwards in the first sliding groove, the position of the nylon net can be raised, the position of the skin piece placed on the nylon net can be raised to the air-liquid level position to be continuously cultivated, and the mode is simple to operate, and the defect that the operation process is complicated in the traditional technology is solved.
For the invention patent with the patent number of US20070181490A1, a preparation method of a PAMPA model is disclosed.
For the invention patent with the patent number of CN 201510818671.3, an artificial skin membrane of liposome is disclosed, the main component of the artificial skin membrane is lecithin, and the preparation process comprises the steps of liposome preparation, sealing of a porous membrane and a nested bottom, combination of the liposome and the porous membrane, freeze thawing cycle and the like.
For the invention patent of CN 200980152302.8, a skin horny layer intercellular lipid mimic substrate prepared by using ceramide, palmitic acid and cholesterol is disclosed, and a lipid membrane formed on the substrate is similar to the lipid lamellar structure of horny layer.
For the patent of the invention with publication number of US10473574, a preparation method of a lipid bionic barrier PermeappaTM is disclosed, the bionic barrier is composed of 2-4 layers of mixture of phospholipid and additive and a hydrated fiber supporting layer, and the permeability coefficient of a model drug passing through the lipid bionic barrier model has better correlation with that of a Caco-2 model and a PAMPA model.
Summarizing: the above related patents have the following problems:
1. the cost of the adopted materials such as lipid is high.
2. The preparation process is complex.
3. Are not used for active substance permeation prediction studies.
4. The lipid biomimetic barrier PermeapadTM is more suitable for simulating the process of in vivo absorption.
On the basis, the invention provides the preparation of the active biological material of the composite stem cells and the application of the active biological material in artificial skin to solve the problem.
Disclosure of Invention
Aiming at the situation, in order to overcome the defects of the prior art, the invention provides the preparation of the active biological material of the composite stem cells and the application of the active biological material in artificial skin, and effectively solves the problems that the existing method is high in manufacturing cost, complex in manufacturing process and not used for active substance research.
The active biological material of the composite stem cells is characterized by comprising a composite material with a double-layer structure, wherein the lower layer is an active material layer which is formed by active liposome and stem cells and is in a hydrogel shape, and the upper layer is an antibacterial layer.
The preparation method of the active biological material of the composite stem cells is characterized by comprising the following steps of:
1) Soybean phospholipids and cholesterol were mixed according to 5:2, dissolving the materials in absolute ethyl alcohol in a mass ratio, placing the dissolved materials in a vacuum rotary steaming instrument, and spin-drying the materials at 45 ℃ under the condition of 200rpm of rotating speed to form a uniform phospholipid membrane;
2) Adding citric acid solution with pH of 4, maintaining phospholipid concentration at 30mg/ml, hydrating the above phospholipid membrane, and then placing in ultrasonic wave for ultrasonic dispersion uniformly, 300W,30s opening for 30s closing, and ultrasonic 1 min every 6ml liposome;
3) Transferring the liquid into a sodium phosphate solution with the concentration of 100mM, and dialyzing for 24 hours by using a dialysis bag with the molecular weight cutoff of 3500Da to obtain the required blank liposome without the inner wrapper;
4) Preparation of liposomes encapsulating active factors: keeping the concentration of the active factors at 1mg/ml, dissolving the active factors in PBS (phosphate buffer solution) at 1mg/ml under the condition of the concentration of phospholipids in a total system at 30mg/ml, adding the dissolved active factors into blank liposome, incubating the blank liposome in a water bath shaking table at 55 ℃ for 1 hour, and dialyzing the blank liposome in a dialysis bag with the molecular weight cutoff of 3500Da at the temperature of 4 ℃ for 24 hours to obtain the liposome encapsulating the active factors;
5) Preparation of polylysine coated active factor liposome: preparing epsilon-polylysine solution with the concentration of 10mg/ml, taking 4ml, dropwise adding encapsulated active peptide liposome under the condition of rotating and stirring at 1200rpm, keeping the rotating and stirring for 1 hour, taking out, centrifuging at 15000rpm in a centrifuge at 4 ℃ for 15 minutes, dissolving with corresponding solvent, and preserving at 4 ℃ to obtain the active liposome.
Preferably, the active factor is derived from marine or terrestrial biological materials, or short peptide active substances from microorganisms produced by genetic engineering techniques, and has a molecular weight of 2KD to 100KD.
Preferably, the preparation steps of the active material layer are as follows:
1) Firstly, preparing sulfhydryl hyaluronic acid: adding 4g of hyaluronic acid into 1L of distilled water, stirring and dissolving to prepare a uniform solution; adding 0.8g of L-cysteine hydrochloride to the hyaluronic acid solution; adding EDC/NHS, stirring and dissolving in dark, and regulating the pH value of the solution to 4.7 to obtain the grafted modified thiolated hyaluronic acid compound; then sequentially dialyzing for 3 days by using deionized water with the pH of 5, sodium chloride solution with the concentration of 1wt% and deionized water with the pH of 5, and then freeze-drying in a dark place to obtain the sulfhydryl hyaluronic acid;
2) The preparation of the composite hydrogel is as follows: taking a proper amount of encapsulated active liposome solution, carrying out ultrasonic treatment, adding 4% by mass of thiolated hyaluronic acid into a stirrer, and adding 0.02% by mass of alpha-ketoglutarate and 1% by mass of N-hydroxysuccinimide compound into the solution after dissolution; gradually dripping 1M sodium hydroxide solution at 1000rpm stirring speed, adjusting pH to 7-8, taking out, placing into 37 deg.C water bath for gelling, and sealing gel in 4 deg.C refrigerator;
3) Again, the preparation of the active peptide-containing liposome hydrogel loaded with stem cells: taking a proper amount of liposome solution for encapsulating active factors, adding 4% of thiolated hyaluronic acid by mass fraction through a stirrer, and adding 0.02% of alpha-ketoglutarate and 1% of N-hydroxysuccinimide compound after dissolution; filtering with 0.22um filter head, sterilizing by ultraviolet irradiation, gradually dripping sterile 1M sodium hydroxide solution, regulating system to neutrality, placing in carbon dioxide incubator at 37deg.C for 10 min, and stabilizing to gel; taking out, adding PBS solution containing 0.1% of the three antibodies to wash the gel system, and then placing the gel system in a carbon dioxide incubator with the temperature of 37 ℃ for incubation for 30min; taking out, adding special culture medium for stem cells, and purifying for 5-7 times; taking stem cells cultured to the third generation, and adjusting the number of the cells to 10 4 And (3) sucking 1ml of stem cell suspension per milliliter, adding the suspension to the surface of gel, putting the gel into a carbon dioxide incubator with the temperature of 37 ℃, and incubating for 24 hours to obtain the liposome hydrogel containing the active peptide and carrying the stem cells.
Preferably, the antibacterial layer is formed by electrostatic spinning of antibacterial material polyhexamethylene biguanide and silk fibroin, and the preparation steps of the antibacterial layer are as follows:
1) Extraction of silk fibroin: adding cut silkworm cocoons into sodium carbonate aqueous solution with the concentration of 0.02M, boiling, degumming until no yellow colloid sediment is generated; washing degummed silk cocoons with a large amount of deionized water, washing, drying to obtain dry silk fibroin, dissolving the dry silk fibroin in a lithium bromide solution with the concentration of 9.3M for 4 hours, dialyzing and freeze-drying to obtain freeze-dried silk fibroin;
2) Preparation of silk fibroin/polyhexamethylene biguanide electrostatic spinning solution: dissolving freeze-dried silk fibroin into spinning solution by using Hexafluoroisopropanol (HFIP), weighing a certain mass of polyhexamethylene biguanide, and dissolving the polyhexamethylene biguanide into the spinning solution at a rotating speed of 120rpm, wherein the mass volume ratio of the polyhexamethylene biguanide in the electrostatic spinning solution is 0.3-1wt%;
3) Preparation of silk fibroin/polyhexamethylene biguanide electrospun membrane: placing the spinning solution in an injector equipped with an 18G needle head, placing on an electrostatic spinning machine, electrospinning under the condition that the voltage is 23KV and the receiving distance is 10-12cm, drying the spun sample in a constant temperature vacuum drying oven, and removing the non-volatile solvent to obtain the antibacterial material layer.
Preferably, the preparation method of the composite material with the double-layer structure comprises the following steps: and compounding an electrostatic spinning antibacterial material layer containing silk fibroin/polyhexamethylene biguanide with liposome hydrogel containing sulfhydrylation hyaluronic acid and encapsulating active factors during gel forming by adopting a Michael addition reaction technology, wherein an electrostatic spinning membrane is used as an upper layer of the double-layer composite material, and the active liposome hydrogel is used as a lower layer, further the upper and lower layers are planted with fibroblasts and adipose-derived stem cells, and the active biological material of the composite stem cells is obtained through gas-liquid co-culture.
Preferably, the stem cells are one of adipose stem cells, umbilical cord stem cells and bone marrow mesenchymal stem cells, or derived from a patient, or derived from a terrestrial animal.
The application of the active biological material of the composite stem cells in the artificial skin is characterized in that the active biological material of the composite stem cells is applied to the artificial skin.
Compared with the prior art, the invention has the following technical effects:
(1) The preparation method adopted by the preparation method of the thiolated hyaluronic acid/H active factor composite hydrogel is mild, a cross-linking agent is not required to be added, and the thiolated hyaluronic acid/H active factor composite hydrogel contains a large amount of active ingredients, and by utilizing the mechanism, the environment can induce angiogenesis through endothelial cells/adipose-derived stem cells combined with active factors and platelet lysate and quickly construct micro-tissues in vitro, so that the scaffold can be beneficial to culturing artificial skin basal layers in vitro on a large scale to meet the demands of patients.
(2) The raw material adopted by the invention has pure natural components, low toxicity and excellent biocompatibility, and is beneficial to cell growth.
(3) The substrate of the invention selects hyaluronic acid, active factor liposome, silk fibroin and polyhexamethylene biguanide to play roles in promoting blood vessels, can quickly construct micro tissues in vitro, is degradable, antibacterial and tensile, can be used as a biological material of a double-layer bionic artificial skin scaffold to be applied to large-area skin defects and the like, and selects hyaluronic acid and active factor liposome to play roles in permeation and absorption, and the loaded active factor liposome and adipose-derived stem cells have main functions of promoting vascularization and proliferation.
(4) The silk fibroin antibacterial film has better strength, bleeding stopping property, antibacterial property and biocompatibility; the upper layer resists the main support material of the membrane and is silk fibroin, so that the antibacterial membrane can guide endothelial cells to grow in an ordered arrangement, and provides certain mechanical strength, biocompatibility, hemostasis, antibacterial and the like.
(5) The upper and lower layers of the artificial skin prepared by the invention can be flexibly combined according to the size and depth of the wound surface, thereby realizing personalized treatment. Wherein the combination of the upper layer and the lower layer of the artificial skin can be realized by Michael addition reaction with the upper antibacterial film in the process of forming the hydrogel.
Drawings
FIG. 1 is a schematic representation of an artificial skin constructed in accordance with the present invention.
FIG. 2 is a microstructure of an artificial skin according to the invention.
FIG. 3 is a graph showing the effect of promoting the growth of blood vessels on the artificial skin according to the invention.
FIG. 4 is a graph showing the effect of the artificial skin animal skin defect treatment of the present invention.
FIG. 5 is a graph showing the antibacterial effect of the antibacterial layer of the present invention.
FIG. 6 is a graph showing the hemostatic effect of the antibacterial layer according to the present invention.
Detailed Description
The foregoing and other features, aspects and advantages of the present invention will become more apparent from the following detailed description of the embodiments with reference to the accompanying drawings, 1-6. The following examples are all referred to in the specification and drawings.
Exemplary embodiments of the present invention will be described below with reference to the accompanying drawings.
The invention aims to overcome the defects of the existing market artificial skin products and provides a double-layer bionic artificial skin bracket and application thereof as a large-area skin defect repair material.
The technical scheme of the invention is as follows:
(1) The hydrogel, the active factors, the stem cells and the antibacterial film are orderly assembled to construct the double-layer bionic artificial skin, so that the technical innovation of repairing the wound surface of the large-area defect is realized.
The natural composite multilevel structure bracket capable of efficiently loading the growth factors is synthesized by adopting the reactions of electrostatic action, disulfide bonds and the like and crosslinking under mild conditions, and the survival rate of stem cells in the hydrogel is improved by providing the pore structure in the bracket and factors such as degraded nutrient substances, mechanical properties, slow-release growth factors and the like, so that a beneficial environment is provided for repairing the large-area skin injury.
(2) The bionic skin active material is utilized to promote the regeneration of blood vessels and reconstruct the regeneration microenvironment, thereby realizing the application innovation of repairing the large-area defect wound surface.
(3) The active factor slow release system is built and added into the hydrogel, so that the function of the active factor slow release system can be utilized to promote the angiogenesis environment in the hydrogel, thereby improving the microenvironment of the sink region, and improving the survival of seed cells in the hydrogel and the adaptability to body tissues.
The active factors and the stem cells are orderly assembled through the hydrogel for the first time, on one hand, the functions of the stem cells are enhanced by utilizing the active liposome hydrogel, the survival and the tolerance to the local micro-environment of transplantation are improved, on the other hand, the active factors are applied to target the host cells, the angiogenesis is promoted, the micro-environment is regenerated again, the treatment effect of wound repair is improved through a double-tube alignment mode, the method has wide clinical application prospect and development transformation potential, and technical support is provided for the repair and regeneration of other complex tissues.
The specific implementation technical means of the invention are as follows:
a double-layer bionic artificial skin scaffold consists of a lower layer-sulfhydryl hyaluronic acid/active factor/adipose stem cell composite hydrogel and an upper layer antibacterial membrane-silk fibroin/polyhexamethylene biguanide electrostatic spinning membrane.
Wherein, the preparation of the lower layer bracket comprises the following steps:
(1) Preparation of active factor liposome: the soybean lecithin (PC) and Cholesterol (CHOL) are dissolved according to a certain proportion to prepare a synthetic liposome which is used for encapsulating small molecule active substances. The dissolved phospholipid and cholesterol were dried by spin drying at 45℃and 200rpm on a vacuum spin-steamer. After a certain volume of citric acid solution (ph=4) was added to hydrate the above phospholipid membrane, the membrane was placed in cell ultrasound for ultrasonic dispersion uniformly and then transferred into sodium phosphate solution (PBS) of a proper concentration, and dialyzed for 24 hours with a dialysis bag of 3500Da molecular weight. After the dialysis is finished, a small amount of sample is left as blank liposome (K-lip), the active factor H and the active factor FITC-H which are dissolved by PBS and have a certain concentration are added into a blank liposome solution to be incubated for 1 hour in a water bath shaker at 55 ℃ (the concentration of each group of drugs is kept to be 1mg/ml, the concentration of the total system phospholipid is kept to be 30 mg/ml), and finally the liposome with the total volume of 15ml is obtained. After removal, the groups were placed in labeled dialysis bags and dialyzed at 4℃for 24h. (dialysis membrane cut-off molecular weight 3500 Da.);
(2) Preparation of polylysine-coated active factor liposome (H-Lip- ε -PL): dissolving epsilon-polylysine (epsilon-PL) with the concentration of 10mg/ml in a flat bottom glass bottle, split charging into 4 ml/bottle, adding magnetic stirring rods into three groups of bottles, placing the bottles on a common multi-channel magnetic stirrer, rotating and stirring at 1200rpm, respectively sucking the blank liposome (K-lip), the active short peptide liposome (H-lip) and the active short peptide liposome (Fitc-H-lip) prepared in the step (1) into 10ml syringes, respectively dropwise adding each group of liposome into the three stirred groups of bottles, keeping the rotating speed for stirring for 1 hour, respectively sucking the three groups of liposomes into a clean EP tube, centrifuging at 15000rpm in a centrifuge at 4 ℃ for 15 minutes, and taking out each group of sucked and removed supernatant, and then resuspending the obtained liposome;
(3) Preparation of a thiol hyaluronic acid/active factor liposome/adipose stem cell composite hydrogel:
(1) modification of thiolated hyaluronic acid: adding hyaluronic acid into distilled water, stirring and dissolving until the solid is completely dissolved to obtain hyaluronic acid solution; adding L-cysteine hydrochloride into a hyaluronic acid solution, adding EDC/NHS, stirring and dissolving in dark place, adjusting the pH value of the solution to 4.7, and grafting the L-cysteine hydrochloride to obtain a sulfhydryl hyaluronic acid compound; then sequentially dialyzing for 3 days with deionized water with pH of 5, deionized water with pH of 5 containing 1wt% of NaCl and deionized water with pH of 5, and then freeze-drying in dark to obtain sulfhydryl hyaluronic acid;
(2) preparation of composite hydrogel: the method is characterized in that the method is used for placing active factor liposome solution under a magnetic stirrer for stirring so as to dissolve a certain proportion of thiolated hyaluronic acid, alpha-ketoglutaric acid and N-hydroxysuccinimide which are added into a specific system volume are used as gel precursor liquid after the dissolution is finished, 1M sodium hydroxide (NaOH) is used for gradually dropwise adding the precursor liquid at a stirring speed of 1000rpm to adjust the pH value of the system to 7-8, the precursor liquid is immediately taken out and separated into templates, the templates are placed into a water bath pot at 37 ℃, and the system is sealed and placed into a refrigerator at 4 ℃ for standby after the system is gelled.
(3) Preparation of hydrogel loaded with fat Stem cells containing active peptide liposomes (ADSCs/H-lip/ε -PL/HASH): preparing a simple gel precursor solution containing active peptide liposome (DDS) by the steps, filtering the gel precursor solution in an ultra-clean bench, filtering the gel precursor solution by a filter head of 0.22um, sterilizing the gel precursor solution by ultraviolet irradiation, gradually dripping 1M sodium hydroxide (NaOH) subjected to sterile filtration into the precursor solution to be neutral, placing the gel precursor solution in a 37 ℃ carbon dioxide incubator for 10 minutes, taking out the gel precursor solution, adding a three-antibody PBS (PBS) containing 0.1% into a sample, slightly and repeatedly soaking the gel system, placing the gel precursor solution in the 37 ℃ carbon dioxide incubator for incubation for 30 minutes, taking out the gel precursor solution, adding a special culture medium for stem cells, purifying the gel precursor solution for 5-7 times, placing the gel precursor solution in the ultra-clean bench for standby, culturing the fat stem cells until the number of the third-generation fat stem cells is adjusted to be 10-4 per milliliter, and taking 1ml of each of the fat stem cell suspension, adding the gel surface, placing the gel solution into the carbon dioxide incubator for incubation for 1 day.
The preparation of the silk fibroin/polyhexamethylene biguanide electrostatic spinning film on the upper layer of the bracket comprises the following steps:
(1) Extraction of silk fibroin: adding cut silkworm cocoons into 0.02M sodium carbonate aqueous solution, boiling, degumming until no yellow colloid sediment is generated; and then washing degummed cocoons with a large amount of deionized water, washing, drying to obtain dry silk fibroin, dissolving the dry silk fibroin in a saturated 9.3M lithium bromide solution for 4 hours, dialyzing and freeze-drying to obtain silk fibroin.
(2) Preparation of silk fibroin/polyhexamethylene biguanide electrostatic spinning solution: the obtained freeze-dried Silk Fibroin (SF) is dissolved into a spinning solution by using Hexafluoroisopropanol (HFIP), a certain mass (PHMB) is weighed, and the solution is dissolved into the silk fibroin spinning solution at a rotating speed of 120rpm, wherein the mass volume ratio of the polyhexamethylene biguanide in the electrostatic spinning solution is 0.3wt% and 1wt%, and the mass volume ratio is named SF/PH0.3 and SF/PH1.
(3) Preparation of silk fibroin/polyhexamethylene biguanide electrospun membrane: transferring the spinning solution prepared in advance into an injector equipped with an 18G needle, placing the injector on an electrostatic spinning machine, applying high voltage of 23KV to a syringe pump to be equipped with a proper push-down matched high-voltage electric field, adjusting the receiving distance to be 10-12cm, fixing a layer of tinfoil on the surface of a receiving rotating shaft to receive spinning, taking off the spun sample together with the tinfoil, drying in a constant-temperature vacuum drying oven, removing non-volatile solvent, and preserving the sample for later use.
Synthesis of double-layer bionic artificial skin scaffold:
the silk fibroin/polyhexamethylene biguanide electrostatic spinning membrane is compounded with the sulfhydrylation hyaluronic acid/H active factor composite hydrogel in the gel forming process through a Michael addition reaction, the silk fibroin/polyhexamethylene biguanide electrostatic spinning membrane is used as the upper layer of the double-layer bionic artificial skin scaffold, the lower layer of the sulfhydrylation hyaluronic acid/H active factor composite hydrogel is respectively planted into the upper layer and the lower layer, the fibroblast and the adipose-derived stem cells are combined through gas-liquid co-culture, and the double-layer scaffold can also be used for layered culture of wound surfaces with different depths and injury areas.
The specific embodiment is as follows:
the preparation method of the artificial skin upper layer-antibacterial film comprises the following steps:
(1) Preparation of spinning solution: adding cut silkworm cocoons into sodium carbonate aqueous solution with the concentration of 0.02M, boiling, degumming until no yellow colloid sediment is generated; washing degummed silk cocoons with a large amount of deionized water, washing, drying to obtain dry silk fibroin, dissolving the dry silk fibroin in a lithium bromide solution with the concentration of 9.3M for 4 hours, dialyzing and freeze-drying to obtain freeze-dried silk fibroin. Dissolving freeze-dried silk fibroin into spinning solution by using Hexafluoroisopropanol (HFIP), weighing a certain mass of polyhexamethylene biguanide, and dissolving into the spinning solution at a rotating speed of 120 rpm. The mass volume ratio of the polyhexamethylene biguanide in the electrostatic spinning solution is 0.3-1wt%.
(2) Preparation of an antibacterial film: preparation of silk fibroin/polyhexamethylene biguanide electrospun membrane: placing the spinning solution prepared in the step (1) in an injector equipped with an 18G needle, placing the injector on an electrostatic spinning machine, carrying out electrospinning under the condition that the voltage is 23KV and the receiving distance is 10-12cm, and drying the spun sample in a constant-temperature vacuum drying oven to remove the non-volatile solvent, thereby obtaining the antibacterial material layer.
Characterization of the properties:
the major diameter of the antibacterial film is distributed about 1um, so that the antibacterial film can be used for cell growth and has an excellent microstructure, as shown in figure 2. In addition, the antibacterial film has excellent antibacterial performance and hemostatic performance and good mechanical performance. The composition has excellent hemostatic effect in the model of large skin hemorrhage and liver hemorrhage of rats, and can be used as a first-aid treatment material for burns and acute wounds. In addition, the material exhibits excellent tissue healing ability and produces more collagen deposition in full-thickness skin infected wounds and is capable of promoting wound closure rates.
Preparation method of active factor liposome
(1) Soybean phospholipids and cholesterol were mixed according to 5:2, dissolving the materials in absolute ethyl alcohol in a mass ratio, placing the dissolved materials in a vacuum rotary steaming instrument, and spin-drying the materials at 45 ℃ under the condition of 200rpm of rotating speed to form a uniform phospholipid membrane;
(2) Adding a certain volume of citric acid solution with pH of 4, keeping phospholipid concentration at 30mg/ml, hydrating the phospholipid membrane, then placing in ultrasonic wave for uniform ultrasonic dispersion, opening 30s for 30s, and performing ultrasonic treatment for 1 min every 6ml of liposome;
(3) Transferring the liquid into a sodium phosphate solution with the concentration of 100mM, and dialyzing for 24 hours by using a dialysis bag with the molecular weight cutoff of 3500Da to obtain the required blank liposome without the inner wrapper;
(4) Preparation of liposomes encapsulating active peptides: the concentration of the active factors is kept at 1mg/ml, under the condition that the concentration of phospholipids in the total system is 30mg/ml, PBS is used for dissolving the active factors at 1mg/ml and is added into blank liposome, after the blank liposome is incubated for 1 hour in a water bath shaking table at 55 ℃, the blank liposome is placed in a dialysis bag with the molecular weight cutoff of 3500Da, and the encapsulated active liposome is obtained after dialysis for 24 hours at the temperature of 4 ℃.
(5) Preparation of polylysine coated active factor liposome: preparing epsilon-polylysine solution with the concentration of 10mg/ml, taking 4ml, dropwise adding encapsulated active peptide liposome under the condition of rotating and stirring at 1200rpm, and keeping stirring at the rotating speed for 1 hour. Taking out, centrifuging in a centrifuge at 4 ℃ at a speed of 15000rpm for 15 minutes, dissolving in a corresponding solvent, and preserving at 4 ℃ to obtain the liposome.
Characterization of the properties:
as can be seen from fig. 3: the H-LIP-epsilon-PL liposome has excellent angiogenesis promoting effect, can promote tubule formation and generate a large number of new capillaries within 24 hours, and the active factors can promote angiogenesis, and slow release the active factors and maintain the activity of the growth factors.
Preparation method of artificial skin lower layer-bioactive hydrogel
(1) Preparation of modified hyaluronic acid:
adding 4g of hyaluronic acid into 1L of distilled water, stirring and dissolving to prepare a uniform solution; adding 0.8g of L-cysteine hydrochloride to the hyaluronic acid solution; adding EDC/NHS, stirring and dissolving in dark, and regulating the pH value of the solution to 4.7 to obtain the grafted modified thiolated hyaluronic acid compound; then sequentially dialyzing for 3 days by using deionized water with the pH of 5, sodium chloride solution with the concentration of 1wt% and deionized water with the pH of 5, and then freeze-drying in a dark place to obtain the sulfhydryl hyaluronic acid;
(2) The preparation of the composite hydrogel is as follows: taking a proper amount of liposome solution for encapsulating active factors, carrying out ultrasonic treatment, adding 4% by mass of thiolated hyaluronic acid into a stirrer, and adding 0.02% by mass of alpha-ketoglutarate and 1% by mass of N-hydroxysuccinimide compound after dissolution; gradually dripping 1M sodium hydroxide solution at 1000rpm stirring speed, adjusting pH to 7-8, taking out, and placing into 37 deg.C water bath for gelling. The gel was then sealed in a refrigerator at 4 ℃ for use.
(3) Again, the preparation of the active peptide-containing liposome hydrogel loaded with stem cells: taking a proper amount of liposome solution for encapsulating active factors, adding 4% of thiolated hyaluronic acid by mass fraction through a stirrer, and adding 0.02% of alpha-ketoglutarate and 1% of N-hydroxysuccinimide compound after dissolution; filtering with 0.22um filter head, sterilizing by ultraviolet irradiation, gradually dripping sterile 1M sodium hydroxide solution, regulating system to neutrality, placing in carbon dioxide incubator at 37deg.C for 10 min, and stabilizing to gel; taking out, adding PBS solution containing 0.1% of the three antibodies to wash the gel system, and then placing the gel system in a carbon dioxide incubator with the temperature of 37 ℃ for incubation for 30min; taking out, adding special culture medium for stem cells, and purifying for 5-7 times; taking stem cells cultured to the third generation, and adjusting the number of the cells to 10 4 And (3) sucking 1ml of stem cell suspension per milliliter, adding the suspension to the surface of gel, putting the gel into a carbon dioxide incubator with the temperature of 37 ℃, and incubating for 24 hours to obtain the liposome hydrogel containing the active peptide and carrying the stem cells.
Characterization of the properties:
the active factor liposome coated by epsilon-polylysine and with the pore diameter of about 100 μm in the water gel shown in figure 2 is uniformly distributed on the inner wall of the gel bracket due to the electrostatic effect to slowly release the active factor;
From the comparison of the groups in FIG. 4, the fluorescent staining results of HE and Masson and CD31 of the combination treatment group show that the combination treatment group-stem cell-loaded composite hydrogel group (ADSCs/CH 02-lip/epsilon-PL/HASH) has the strongest tissue healing capacity, generates a large number of new blood vessels at the defect site, has the best collagen deposition condition and can promote the wound closure rate. Therefore, the treatment effect of the composite hydrogel group loaded with stem cells (ADSCs/CH 02-lip/epsilon-PL/HASH) is superior to that of other experimental groups. The bioactive hydrogel has the characteristics of low immunogenicity and high biocompatibility.
The preparation method of the double-layer artificial skin scaffold comprises the following steps:
(1) Wetting and incubating the fully dried antibacterial film with the composite hydrogel precursor solution for one hour at 37 ℃, placing the prepared composite hydrogel on the surface of the wetted antibacterial film, soaking the whole material in a culture dish containing the precursor solution, adjusting the precursor solution to be neutral by using 1M sodium hydroxide, and then placing the culture dish in the temperature of 37 ℃ for incubation for 1 hour to form the double-layer artificial skin stent-antibacterial film/bioactive hydrogel.
(2) The scaffold can be used for culturing the bionic dermis layer epidermis layer cell-containing scaffold on a large scale through a gas-liquid co-culture aliquoting layer culture environment, active factors used for the cell culture environment are all from mild active factors and platelet lysate which are extracted autonomously, and dermis layer cells are derived from co-cultures of adipose-derived stem cells and endothelial cells of patients, so that the scaffold has the characteristics of low immunogenicity and high biocompatibility.
Characterization of the properties:
after freeze-drying the material, observing the material through a scanning electron microscope to find that the upper layer of the scaffold is a nanofiber electrostatic spinning scaffold with the diameter of about 1 mu m, the pore diameter of the hydrogel at the lower layer is about 100 mu m, and the epsilon-polylysine coated active factor liposome is uniformly distributed on the inner wall of the gel scaffold due to the electrostatic effect to slowly release the active factor; cross-linking occurs between the two material interfaces, see figure 2. The double-layer material can be prepared according to the actual condition of a patient, realizes personalized treatment, and is expected to become an artificial skin bracket material with great potential.
The preparation method is mild, natural and nontoxic, is beneficial to cell growth, can quickly construct micro-tissues in vitro, has the functions of degradability, antibiosis, tension and the like, and has better strength, hemostatic property, antibiosis and biocompatibility.
While the present invention has been described in detail by the foregoing description of the preferred embodiment, it should be understood that the above description should not be construed as limiting the invention, but rather as many modifications and substitutions will become apparent to those skilled in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.
Claims (8)
1. The active biological material of the composite stem cells is characterized by comprising a composite material with a double-layer structure, wherein the lower layer is an active material layer which is formed by active liposome and stem cells and is in a hydrogel shape, and the upper layer is an antibacterial layer.
2. The method for preparing an active biological material of a composite stem cell according to claim 1, wherein the preparation steps of the active liposome are as follows:
1) Soybean phospholipids and cholesterol were mixed according to 5:2, dissolving the materials in absolute ethyl alcohol in a mass ratio, placing the dissolved materials in a vacuum rotary steaming instrument, and spin-drying the materials at 45 ℃ under the condition of 200rpm of rotating speed to form a uniform phospholipid membrane;
2) Adding citric acid solution with pH of 4, maintaining phospholipid concentration at 30mg/ml, hydrating the above phospholipid membrane, and then placing in ultrasonic wave for ultrasonic dispersion uniformly, 300W,30s opening for 30s closing, and ultrasonic 1 min every 6ml liposome;
3) Transferring the liquid into a sodium phosphate solution with the concentration of 100mM, and dialyzing for 24 hours by using a dialysis bag with the molecular weight cutoff of 3500Da to obtain the required blank liposome without the inner wrapper;
4) Preparation of liposomes encapsulating active factors: keeping the concentration of the active factors at 1mg/ml, dissolving the active factors in PBS (phosphate buffer solution) at 1mg/ml under the condition of the concentration of phospholipids in a total system at 30mg/ml, adding the dissolved active factors into blank liposome, incubating the blank liposome in a water bath shaking table at 55 ℃ for 1 hour, and dialyzing the blank liposome in a dialysis bag with the molecular weight cutoff of 3500Da at the temperature of 4 ℃ for 24 hours to obtain the liposome encapsulating the active factors;
5) Preparation of polylysine coated active factor liposome: preparing epsilon-polylysine solution with the concentration of 10mg/ml, taking 4ml, dropwise adding encapsulated active peptide liposome under the condition of rotating and stirring at 1200rpm, keeping the rotating and stirring for 1 hour, taking out, centrifuging at 15000rpm in a centrifuge at 4 ℃ for 15 minutes, dissolving with corresponding solvent, and preserving at 4 ℃ to obtain the active liposome.
3. The method for preparing an active biological material of composite stem cells according to claim 2, wherein the active factor is derived from marine biological material, terrestrial biological material, or a short peptide active substance derived from microorganism, which is manufactured by genetic engineering technology, and has a molecular weight of 2KD to 100KD.
4. The method for preparing an active biological material of a composite stem cell according to claim 2, wherein the step of preparing the active material layer comprises the steps of:
1) Firstly, preparing sulfhydryl hyaluronic acid: adding 4g of hyaluronic acid into 1L of distilled water, stirring and dissolving to prepare a uniform solution; adding 0.8g of L-cysteine hydrochloride to the hyaluronic acid solution; adding EDC/NHS, stirring and dissolving in dark, and regulating the pH value of the solution to 4.7 to obtain the grafted modified thiolated hyaluronic acid compound; then sequentially dialyzing for 3 days by using deionized water with the pH of 5, sodium chloride solution with the concentration of 1wt% and deionized water with the pH of 5, and then freeze-drying in a dark place to obtain the sulfhydryl hyaluronic acid;
2) The preparation of the composite hydrogel is as follows: taking a proper amount of encapsulated active liposome solution, carrying out ultrasonic treatment, adding 4% by mass of thiolated hyaluronic acid into a stirrer, and adding 0.02% by mass of alpha-ketoglutarate and 1% by mass of N-hydroxysuccinimide compound into the solution after dissolution; gradually dripping 1M sodium hydroxide solution at 1000rpm stirring speed, adjusting pH to 7-8, taking out, placing into 37 deg.C water bath for gelling, and sealing gel in 4 deg.C refrigerator;
3) Again, the preparation of the active peptide-containing liposome hydrogel loaded with stem cells: taking a proper amount of liposome solution for encapsulating active factorsAdding 4% by mass of thiolated hyaluronic acid through a stirrer, and adding 0.02% by mass of alpha-ketoglutaric acid and 1% by mass of N-hydroxysuccinimide compound after dissolution; filtering with 0.22um filter head, sterilizing by ultraviolet irradiation, gradually dripping sterile 1M sodium hydroxide solution, regulating system to neutrality, placing in carbon dioxide incubator at 37deg.C for 10 min, and stabilizing to gel; taking out, adding PBS solution containing 0.1% of the three antibodies to wash the gel system, and then placing the gel system in a carbon dioxide incubator with the temperature of 37 ℃ for incubation for 30min; taking out, adding special culture medium for stem cells, and purifying for 5-7 times; taking stem cells cultured to the third generation, and adjusting the number of the cells to 10 4 And (3) sucking 1ml of stem cell suspension per milliliter, adding the suspension to the surface of gel, putting the gel into a carbon dioxide incubator with the temperature of 37 ℃, and incubating for 24 hours to obtain the liposome hydrogel containing the active peptide and carrying the stem cells.
5. The method for preparing an active biological material of a composite stem cell according to claim 4, wherein the antibacterial layer is formed by electrostatic spinning of an antibacterial material polyhexamethylene biguanide and silk fibroin, and the preparation steps of the antibacterial layer are as follows:
1) Extraction of silk fibroin: adding cut silkworm cocoons into sodium carbonate aqueous solution with the concentration of 0.02M, boiling, degumming until no yellow colloid sediment is generated; washing degummed silk cocoons with a large amount of deionized water, washing, drying to obtain dry silk fibroin, dissolving the dry silk fibroin in a lithium bromide solution with the concentration of 9.3M for 4 hours, dialyzing and freeze-drying to obtain freeze-dried silk fibroin;
2) Preparation of silk fibroin/polyhexamethylene biguanide electrostatic spinning solution: dissolving freeze-dried silk fibroin into spinning solution by using Hexafluoroisopropanol (HFIP), weighing a certain mass of polyhexamethylene biguanide, and dissolving the polyhexamethylene biguanide into the spinning solution at a rotating speed of 120rpm, wherein the mass volume ratio of the polyhexamethylene biguanide in the electrostatic spinning solution is 0.3-1wt%;
3) Preparation of silk fibroin/polyhexamethylene biguanide electrospun membrane: placing the spinning solution in an injector equipped with an 18G needle head, placing on an electrostatic spinning machine, electrospinning under the condition that the voltage is 23KV and the receiving distance is 10-12cm, drying the spun sample in a constant temperature vacuum drying oven, and removing the non-volatile solvent to obtain the antibacterial material layer.
6. The method for preparing an active biological material of a composite stem cell according to claim 5, wherein the method for preparing the composite material with a double-layer structure comprises the following steps: and compounding an electrostatic spinning antibacterial material layer containing silk fibroin/polyhexamethylene biguanide with liposome hydrogel containing sulfhydrylation hyaluronic acid and encapsulating active factors during gel forming by adopting a Michael addition reaction technology, wherein an electrostatic spinning membrane is used as an upper layer of the double-layer composite material, and the active liposome hydrogel is used as a lower layer, further the upper and lower layers are planted with fibroblasts and adipose-derived stem cells, and the active biological material of the composite stem cells is obtained through gas-liquid co-culture.
7. The method of claim 4, wherein the stem cells are adipose stem cells, umbilical cord stem cells, or bone marrow mesenchymal stem cells, or derived from a patient's own body, or derived from a terrestrial animal.
8. Use of an active biological material of composite stem cells according to any one of claims 1-7 in artificial skin, wherein the active biological material of composite stem cells is used in artificial skin.
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