Preparation of a composite stem cell active biomaterial and its application in artificial skin

Abstract

The application is a preparation of a composite stem cell active biomaterial and its application in artificial skin, effectively solves the problems of high production cost, complex production process and non-use for active substance research of the existing method, and comprises a double-layer composite material, the lower layer is an active material layer in the form of hydrogel composed of active liposomes and stem cells, and the upper layer is an antibacterial layer; the preparation method of the thiolated hyaluronic acid / H active factor composite hydrogel is relatively mild, does not need to add a crosslinking agent and contains a large amount of active ingredients; by using the mechanism, the environment can induce angiogenesis through endothelial cells / fat stem cells combined with active factors and platelet lysate and rapidly construct microtissues in vitro, which is beneficial to the scaffold in large-scale cultivation of artificial skin basement layer in vitro to meet the needs of patients.

Description

Technical Field

[0001] This invention relates to the field of biomimetic polymer materials technology, specifically to the preparation of a composite stem cell active biomaterial and its application in artificial skin. Background Technology

[0002] Understanding: The skin is the largest organ in the human body, playing a vital protective and physiological role. However, severe skin damage caused by burns, trauma, and intractable skin ulcers (such as diabetic foot) is a major medical and public health problem faced by countries worldwide. In clinical practice, the repair of difficult-to-heal wounds and large-area trauma requires the grafting of natural or artificial skin onto their surface.

[0003] Although natural skin (including autologous skin, allogeneic skin, and xenogeneic skin) has a structure and function similar to that of the recipient skin, it has problems such as insufficient skin source, immune rejection, and viral transmission, thus limiting its application in the repair of large-area wounds.

[0004] Artificial skin refers to skin substitutes artificially developed in vitro using principles and methods of engineering and cell biology to repair or replace damaged skin tissue. Artificial skin closely resembles human skin and is highly effective in treating burns and scalds by reducing pain and preventing scarring. It also shows good efficacy in treating chronic, intractable ulcers such as diabetic foot. Internationally commercially available artificial skin with dermal replacement function uses animal-derived biomaterials similar to human skin. However, due to the xenogeneic nature of these biomolecules, they can still induce a certain degree of immune rejection (such as inflammatory reactions) after transplantation, affecting rapid wound healing. Furthermore, large-area transplantation carries the risk of inducing systemic inflammatory response syndrome.

[0005] A truly effective artificial skin should possess the three-dimensional structure of native skin and perform the functions of natural skin tissue. Furthermore, it should support angiogenesis and provide support for cells present in the local environment. After implantation, it must also be able to integrate with the host with minimal scarring while generating a controlled inflammatory response. Large-area full-thickness skin defects are a challenging problem in wound care worldwide, and sudden trauma, accidents, and other incidents can have a significant impact on human health and quality of life. The development of stem cell and tissue engineering has revolutionized the treatment of tissue damage and organ failure, triggering a new medical revolution following drugs and surgery. Currently, autologous skin transplantation is widely used as an important treatment for full-thickness skin defects, with spironolactone being one of the earliest representative products. However, due to insufficient vascularization or skin transplantation failure, wound healing may be delayed, increasing the risk of infection, and the formation of skin appendages is rare. In addition, wounds infected by microorganisms and blood supply disruptions caused by vascular defects are among the main factors leading to complications of full-thickness skin defects. Currently, no single skin substitute on the market has been proven to fully restore normal skin structure and physiological function. It is not comparable to current market products.

[0006] Currently available artificial skin product brands include Dermagraft, Integra, AlloDerm, MatriStem, PriMatrix, PELNAC, and PermeaDerm skin dressings; as well as tissue-engineered composite skins (artificial skin) such as EpiSkin, EpiDerm, MatriDerm, Apligraf, and ActivSkin.

[0007] Search: A search was conducted in the patent database for this relevant field, and dozens of related patents were found. Among them, the following patents are more similar to the technical problem solved by this application.

[0008] Citation:

[0009] The invention patent with publication number CN110960730B discloses a 3D-printed biomimetic anti-rejection artificial skin and its preparation method. The method involves dissolving amino-containing polysaccharides and proteins in an acidic solution to obtain a precursor solution. Spermine and small-molecule dialdehyde or a polymer material modified with aldehyde groups at both ends are dissolved in anhydrous ethanol to obtain a spermine crosslinking agent. A 3D bioprinting method is used to print the precursor solution in a petri dish and atomize the spermine crosslinking agent. This process is repeated sequentially, and the hardness of each layer is controlled by adjusting the concentration of the spermine crosslinking agent in each layer, ultimately resulting in a biomimetic anti-rejection artificial skin with a gradient of hardness.

[0010] The invention patent with publication number CN111228572B discloses an artificial skin and its preparation method and application. The steps include: uniformly coating liquid silicone rubber onto the surface of a solid support, performing a first curing treatment to form an incompletely cured silicone rubber layer; directly placing a pre-formed collagen membrane onto the surface of the incompletely cured silicone rubber layer from step S1, performing a second curing treatment to completely cure the silicone rubber layer and tightly bond it to the collagen membrane; separating the completely cured silicone rubber layer, which has the collagen membrane bonded in the first step, from the solid support to obtain the artificial skin.

[0011] The invention patent with publication number CN111407929A provides a novel artificial skin and its preparation method. The specific preparation method of this novel artificial skin is as follows: Fresh animal skin is taken and treated with a series of agents including sodium hydroxide, DNase, trypsin, sodium dodecyl sulfate, peracetic acid, and PBS to obtain a decellularized dermal matrix. Graphene oxide is then ultrasonically mixed with the decellularized dermal matrix solution, and after enrichment treatment, a novel artificial skin is obtained.

[0012] The invention patent with publication number CN111494713A discloses a micro-nano biomimetic artificial skin repair membrane, comprising a basement membrane, a scaffold layer, an epidermal membrane, and a separable surface membrane; the basement membrane, scaffold layer, polyurethane epidermal membrane, and separable surface membrane are stacked sequentially from the inside out; the basement membrane is a nonwoven membrane made of sericin electrospun; the diameter of the sericin electrospun fibers is 50-500 nm; the scaffold layer is a nonwoven layer made of blended electrospun fibers; the blended electrospun fibers are helical fibers formed by blending PLA and PCL; the diameter of the blended electrospun fibers is 0.2-5 μm; the epidermal membrane is a biodegradable polyurethane film; and the separable surface membrane is woven from polypropylene filaments.

[0013] The invention patent with publication number CN113174138A discloses an artificial skin membrane containing layered liquid crystals, its preparation method, and its application. The artificial skin membrane comprises a substrate material and a lyotropic liquid crystal component. The substrate material is a network structure formed by polydimethylsiloxane and chitosan. The lyotropic liquid crystal component accounts for 3-8% of the mass percentage of the artificial skin membrane. The lyotropic liquid crystal component is selected from one or more of the following: 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.

[0014] The invention patent with publication number CN113105711A provides an artificial skin that simulates perspiration, comprising a water-permeable layer and a temperature-sensitive polyurethane hydrogel layer. The preparation steps include: weighing the carbon nanotubes, polyether polyol, isocyanate, chain extender, N-ethylmorpholine, tantalum pentoxide, pentaerythritol, stannous octoate, and disodium magnesium ethylenediaminetetraacetate according to weight parts; emulsifying to obtain a carbon nanotube emulsion; introducing ozone gas to obtain hydrophilic carbon nanotubes; placing the modified carbon nanotubes, polyether polyol, isocyanate, N-ethylmorpholine, and tantalum pentoxide in a reaction vessel, setting the reaction conditions, and preparing a polyurethane semi-finished product; adding the chain extender, pentaerythritol, and stannous octoate to the reaction vessel to prepare a polyurethane prepolymer; further reacting the polyurethane prepolymer and disodium magnesium ethylenediaminetetraacetate to obtain a temperature-sensitive polyurethane hydrogel.

[0015] The invention patent with publication number CN113577398A discloses a 3D-printed artificial skin and its preparation method. The skin includes an epidermis and a dermis. The epidermis is applied to the upper surface of the dermis and is in a hydrogel state with pH sensitivity. The components of the epidermis include gelatin, tannic acid, and iron salt, which improves the mechanical properties of the epidermis and provides long-lasting and stable antibacterial and anti-inflammatory properties. This solves the problem in the prior art where the explosive release of antibacterial substances from the epidermis causes cytotoxicity to normal cells.

[0016] The invention patent with publication number CN114108177A discloses an artificial skin material capable of photothermally triggering the phased release of growth factors, its preparation method, and its application. It relates to an artificial skin material capable of photothermally triggering the phased release of growth factors and its preparation method. Using biodegradable polymer materials, biodegradable natural polymers, and phase change materials as main raw materials, and adding antibacterial drugs and multiple growth factors, a scaffold material with multi-level repair effects is prepared by electrospinning. Different types of antibacterial drugs are added to the material to achieve multiple antibacterial effects. Phase change material particles containing different types of growth factors are deposited between two fiber layers, enabling photothermal triggering of the spatiotemporally controlled and orderly release of multiple growth factors, providing the microenvironment required for different stages of wound healing.

[0017] The invention patent with announcement number CN113797388 A discloses a chitin artificial skin membrane and its preparation method. By using a low-temperature freeze-thaw method with chitin powder as raw material, the effects of sodium hydroxide concentration, chitin dosage, and urea concentration on membrane material formation are studied. The effects of composite material type and ratio on the mechanical properties of composite membrane are also studied. The preparation conditions are optimized to prepare a multifunctional membrane material with suitable strength, degradation rate, and mechanical properties.

[0018] The invention patent with publication number CN114288474A discloses a dermis layer for promoting hair follicle regeneration, artificial skin, and its preparation method. The raw materials for the dermis layer include collagen, YAP protein inhibitors, and growth factor microspheres. By using YAP protein inhibitors to effectively block the activation of serrated protein genes in wounds, the recovery of secondary skin components (hair follicles and glands), extracellular matrix structure, and tensile strength are indistinguishable from uninjured skin.

[0019] The invention patent with publication number CN114470338A discloses a dermis, artificial skin, and a method for preparing the same. The raw materials for the dermis include recombinant human collagen, verteporfen, and an enzyme. Vertepofen provides an effective mechanotransduction mechanism, regulating the expression of En1 during wound healing and inducing the restoration of normal dermal ultrastructure. This solves the problems of scar formation and the inability to generate hair follicles, sebaceous glands, and other dermal appendages during skin healing.

[0020] The invention patent with publication number CN214881593U discloses a multifunctional artificial skin culture medical device, including a main body. A skin block to be cultured is placed on a nylon mesh. Then, a first rotary knob is rotated, which drives a one-way screw to rotate. The rotation of the one-way screw causes a first slider to move downwards within a first groove, lowering the nylon mesh and allowing the skin block placed on the mesh to be completely immersed in the culture medium, floating for culture. After a period of culture, the first rotary knob is rotated in the opposite direction, causing the one-way screw to rotate in the opposite direction, allowing the first slider to move upwards within the first groove, raising the nylon mesh and raising the skin block placed on the mesh to the air-liquid surface for continued culture. This method is simple to operate and overcomes the cumbersome operation of traditional technologies.

[0021] The invention patent with patent number US20070181490A1 discloses a method for preparing a PAMPA model.

[0022] The invention patent with patent number CN201510818671.3 discloses a liposome artificial skin membrane, the main component of which is lecithin. The preparation process includes steps such as liposome preparation, sealing of the porous membrane and the nested bottom, binding of liposomes and porous membrane, and freeze-thaw cycle.

[0023] The invention patent with patent number CN200980152302.8 discloses a skin stratum corneum intercellular lipid simulation substrate prepared using ceramide, palmitic acid and cholesterol, wherein the lipid membrane formed on the substrate is similar to the lipid layer structure of the stratum corneum.

[0024] The invention patent with publication number US10473574 discloses a method for preparing a lipid biomimetic barrier Permeapad™. The biomimetic barrier is composed of a mixture of 2-4 layers of phospholipids and additives and a hydrated fiber support layer. The permeability coefficient of the model drug through the lipid biomimetic barrier model has a good correlation with the permeability coefficients of the Caco-2 model and the PAMPA model.

[0025] In summary, the above-mentioned patents have the following problems:

[0026] 1. The lipids and other materials used are relatively expensive.

[0027] 2. The preparation process is relatively complex.

[0028] 3. Not used in studies predicting the penetration of active substances.

[0029] 4. The lipid biomimetic barrier Permeapad™ is better suited to mimic the absorption process in vivo.

[0030] Based on this, the present invention provides a method for preparing a composite stem cell biomaterial and its application in artificial skin to solve this problem. Summary of the Invention

[0031] In view of the above situation and to overcome the defects of the prior art, the present invention provides a method for preparing a composite stem cell biomaterial and its application in artificial skin, which effectively solves the problems of high production cost, complex production process and lack of application in the research of active substances in the existing methods.

[0032] A composite stem cell biomaterial is characterized by comprising a bilayer composite material, wherein the lower layer is an active material layer composed of active liposomes and stem cells in a hydrogel-like state, and the upper layer is an antibacterial layer.

[0033] A method for preparing a bioactive biomaterial composed of composite stem cells, characterized in that the preparation step of the bioactive liposomes is as follows:

[0034] 1) Soybean phospholipids and cholesterol were dissolved in anhydrous ethanol at a mass ratio of 5:2. The solution was placed in a vacuum rotary evaporator and evaporated at 45°C and 200 rpm to form a uniform phospholipid film.

[0035] 2) Add citric acid solution with pH 4 to maintain phospholipid concentration at 30 mg / ml, hydrate the above phospholipid membrane, and then place it in an ultrasonic bath to disperse it evenly. Use 300W, turn on for 30 seconds and turn off for 30 seconds, and sonicate each 6 ml of liposomes for 1 minute.

[0036] 3) Transfer the above liquid into a 100mM sodium phosphate solution and dialyze it for 24 hours using a dialysis bag with a molecular weight cutoff of 3500 Da to obtain the desired blank liposomes without inner encapsulation.

[0037] 4) Preparation of liposomes encapsulating active factors: Under the conditions of maintaining the concentration of active factors at 1 mg / ml and the concentration of phospholipids in the total system at 30 mg / ml, the concentration of active factors at 1 mg / ml was dissolved in PBS and added to blank liposomes. After incubating in a water bath shaker at 55°C for 1 hour, the liposomes were dialyzed in a dialysis bag with a molecular weight cutoff of 3500 Da at 4°C for 24 hours to obtain liposomes encapsulating active factors.

[0038] 5) Preparation of polylysine-encapsulated active factor liposomes: Prepare an ε-polylysine solution with a concentration of 10 mg / ml. Take 4 ml of the solution and add the encapsulated active peptide liposomes dropwise while stirring at 1200 rpm. Keep stirring for 1 hour. Remove the solution and centrifuge at 15000 rpm for 15 minutes at 4℃. Dissolve the solution in the appropriate solvent and store at 4℃ to obtain the active liposomes.

[0039] Preferably, the active factor is derived from marine biological materials, terrestrial biological materials, or short peptide active substances from microorganisms manufactured using genetic engineering technology, with a molecular weight between 2KD and 100kD.

[0040] Preferably, the preparation steps of the active material layer are as follows:

[0041] 1) First, the preparation of thiolized hyaluronic acid: 4g of hyaluronic acid was added to 1L of distilled water and stirred to dissolve, thus preparing a homogeneous solution; then 0.8g of L-cysteine ​​hydrochloride was added to the hyaluronic acid solution; EDC / NHS was then added and stirred to dissolve in the dark, and the pH of the solution was adjusted to 4.7 to obtain the grafted modified thiolized hyaluronic acid complex; then, the solution was dialyzed for 3 days each with deionized water at pH 5, sodium chloride solution at 1wt%, and deionized water at pH 5, respectively, and then freeze-dried in the dark to obtain thiolized hyaluronic acid;

[0042] 2) Next is the preparation of the composite hydrogel: After sonicating an appropriate amount of encapsulated active liposome solution, add 4% mercapto-modified hyaluronic acid to the stirrer. After dissolution, add 0.02% α-ketoglutarate and 1% N-hydroxysuccinimide complex. Under a stirring speed of 1000 rpm, gradually add 1M sodium hydroxide solution to adjust the pH of the system to 7-8. Take it out and place it in a 37℃ water bath to form a gel. Then seal the gel and place it in a 4℃ refrigerator for later use.

[0043] 3) Next, the preparation of liposome hydrogels containing active peptides loaded with stem cells: Take an appropriate amount of liposome solution encapsulating active factors and add 4% (w / w) thiolated hyaluronic acid through a stirrer. After dissolution, add a complex of 0.02% (w / w) α-ketoglutarate and 1% (w / w) N-hydroxysuccinimide. Place it in a clean bench, filter it through a 0.22µm filter, sterilize it under UV irradiation, and gradually add sterile 1M sodium hydroxide solution to adjust the system to neutral. Place it in a CO2 incubator at 37℃ for 10 minutes to stabilize the gel. After removal, rinse the gel system with PBS solution containing 0.1% triple antibodies, and then incubate it in a CO2 incubator at 37℃ for 30 minutes. Remove it and add stem cell-specific culture medium to purify it 5-7 times. Take stem cells cultured to the third generation and adjust the cell count to 10-1. 4 1 ml of stem cell suspension was added to the surface of the gel and placed in a carbon dioxide incubator at 37°C for 24 hours to obtain a stem cell-loaded liposome hydrogel containing active peptides.

[0044] Preferably, the antibacterial layer is composed of antibacterial material polyhexamethylene biguanide and silk fibroin through electrospinning, and the preparation steps of the antibacterial layer are as follows:

[0045] 1) Extraction of silk fibroin: Take the cut silkworm cocoons and add them to a 0.02M sodium carbonate aqueous solution. Boil and degumme until no yellow gelatinous precipitate is produced. Wash the degummed silkworm cocoons with a large amount of deionized water, and dry them to obtain dry silk fibroin. Dissolve the dry silk fibroin in a 9.3M lithium bromide solution for 4 hours, dialyze and freeze dry to obtain freeze-dried silk fibroin.

[0046] 2) Preparation of silk fibroin / polyhexamethylbiguanide electrospinning solution: Lyophilized silk fibroin was dissolved in hexafluoroisopropanol (HFIP) to prepare a spinning solution. A certain mass of polyhexamethylbiguanide was weighed and dissolved in the spinning solution at a speed of 120 rpm. The mass-to-volume ratio of polyhexamethylbiguanide in the electrospinning solution was 0.3-1 wt%.

[0047] 3) Preparation of silk fibroin / polyhexamethyl biguanide electrospun membrane: Take the above spinning solution into a syringe equipped with an 18G needle, place it on an electrospinning machine, and electrospin under the conditions of 23KV voltage and receiving distance of 10-12cm. After spinning, dry the sample in a constant temperature vacuum drying oven to remove the unvolatile solvent, and thus obtain the antibacterial material layer.

[0048] Preferably, the preparation method of the bilayer composite material is as follows: using Michael addition reaction technology, an electrospun antibacterial material layer containing silk fibroin / polyhexamethylbiguanide is combined with a liposome hydrogel containing thiolized hyaluronic acid and encapsulating active factors during gelation. The electrospun membrane serves as the upper layer of the bilayer composite material, and the active liposome hydrogel serves as the lower layer. Fibroblasts and adipose-derived stem cells are further seeded in the upper and lower layers, and the active biomaterial of composite stem cells is obtained through gas-liquid co-culture.

[0049] Preferably, the stem cells are one of adipose stem cells, umbilical cord stem cells, or bone marrow mesenchymal stem cells, or are derived from the patient's own body or from terrestrial animals.

[0050] The application of a composite stem cell biomaterial in artificial skin, characterized in that the composite stem cell biomaterial is applied to artificial skin.

[0051] Compared with the prior art, the present invention has the following technical effects:

[0052] (1) The preparation method used in this invention to prepare thiolized hyaluronic acid / H active factor composite hydrogel is relatively mild, does not require the addition of cross-linking agent and contains a large number of active ingredients. Using this mechanism, angiogenesis can be induced by endothelial cells / adipose stem cells in combination with active factors and platelet lysates, and micro-tissues can be rapidly constructed in vitro. This is beneficial for the scaffold to be used to cultivate artificial skin basal layer on a large scale in vitro to meet the needs of patients.

[0053] (2) The raw materials used in this invention are all-natural and low in toxicity, and have excellent biocompatibility, which is beneficial to cell growth.

[0054] (3) In terms of the substrate of the present invention, hyaluronic acid, active factor liposomes, silk fibroin and polyhexamethylene biguanide are selected to promote angiogenesis, rapidly construct micro-tissues in vitro, degrade, antibacterial and anti-tensile effects, and can be used as a biomaterial for a double-layer biomimetic artificial skin scaffold for large-area skin defects. In terms of the substrate, not only are hyaluronic acid and active factor liposomes selected to have the effect of permeability and absorption, but the active factor liposomes and adipose stem cells loaded on it have the main functions of promoting angiogenesis and promoting proliferation.

[0055] (4) The silk fibroin antibacterial membrane obtained by the present invention has good strength, hemostasis, antibacterial and biocompatibility. The main support material of the upper protective membrane is silk fibroin, which enables the antibacterial membrane to guide the orderly arrangement and growth of endothelial cells, and provides certain mechanical strength, biocompatibility and hemostasis and antibacterial properties.

[0056] (5) The upper and lower layers of the artificial skin made in this invention can be flexibly combined according to the size and depth of the wound to achieve personalized treatment. The combination of the upper and lower layers of the artificial skin can be achieved through Michael addition reaction with the upper antibacterial membrane during the hydrogel formation process. Attached Figure Description

[0057] Figure 1 This is a diagram of the artificial skin constructed for this invention.

[0058] Figure 2 This is a microscopic structural diagram of the artificial skin of the present invention.

[0059] Figure 3 This is an image illustrating the effect of artificial skin promoting blood vessel growth according to the present invention.

[0060] Figure 4 This image shows the effect of the artificial skin used in the treatment of skin defects in animals according to the present invention.

[0061] Figure 5 This is a diagram illustrating the antibacterial effect of the antibacterial layer of the present invention.

[0062] Figure 6 This is a diagram illustrating the hemostatic effect of the antibacterial layer of the present invention. Detailed Implementation

[0063] The foregoing and other technical contents, features and effects of the present invention are described in conjunction with the appendix below. Figures 1 to 6 The detailed description of the embodiments will make this clear. All references to the following embodiments are made with reference to the accompanying drawings.

[0064] Exemplary embodiments of the present invention will now be described with reference to the accompanying drawings.

[0065] The purpose of this invention is to address the shortcomings of current artificial skin products on the market and to provide a double-layer bionic artificial skin scaffold and its application as a material for repairing large-area skin defects.

[0066] Invention concept:

[0067] (1) By orderly assembling hydrogel, active factors, stem cells and antibacterial membrane, a double-layer biomimetic artificial skin is constructed to achieve a technological innovation in the repair of large-area defect wounds.

[0068] By employing electrostatic interactions and disulfide bond reactions, a natural composite multi-level scaffold capable of efficiently loading growth factors is synthesized under mild conditions through cross-linking. This improves the survival rate of stem cells in hydrogels by providing internal pore structures, degraded nutrients, mechanical properties, and sustained-release growth factors, thus creating a beneficial environment for the repair of large-area skin damage.

[0069] (2) Utilize biomimetic skin active materials to promote blood vessel regeneration, rebuild the regenerative microenvironment, and achieve innovative applications in the repair of large-area defect wounds.

[0070] (3) An active factor sustained-release system was constructed and added to the hydrogel in order to utilize its own function to promote the angiogenesis environment in the hydrogel, thereby improving the microenvironment of the host area and enhancing the survival of seed cells in the hydrogel and their adaptation to the body tissue.

[0071] For the first time, active factors and stem cells are assembled in an orderly manner using hydrogels. On the one hand, the active liposome hydrogel is used to enhance the function of stem cells, improve their survival and tolerance to the local microenvironment of transplantation. On the other hand, the active factors are applied to target host cells, promote angiogenesis and rebuild the regenerative microenvironment. Through this two-pronged approach, the therapeutic effect of wound repair is improved. This approach not only has broad clinical application prospects and development and translational potential, but also provides technical support for the repair and regeneration of other complex tissues.

[0072] The specific technical means for implementing this invention are as follows:

[0073] A double-layer biomimetic artificial skin scaffold comprises a lower layer of thiolized hyaluronic acid / active factor / adipose stem cell composite hydrogel and an upper layer of antibacterial membrane—silk fibroin / polyhexamethylene biguanide electrospun membrane.

[0074] The preparation of the lower scaffold involves the following steps:

[0075] (1) Preparation of active factor liposomes: Soybean lecithin (PC) and cholesterol (CHOL) were dissolved in a certain ratio to prepare synthetic liposomes for encapsulating small molecule active substances. The dissolved phospholipid and cholesterol solution was placed in a vacuum rotary evaporator and evaporated at 45℃ and 200rpm. A certain volume of citric acid solution (pH=4) was added to hydrate the above phospholipid membrane, and then it was ultrasonically dispersed in a cell sonicator and transferred to an appropriate concentration of sodium phosphate solution (PBS) and dialyzed for 24h using a dialysis bag with a molecular weight of 3500Da. After dialysis, a small amount of sample was reserved as blank liposomes (K-lip). A certain concentration of active factor H and active factor FITC-H dissolved in PBS were added to the blank liposome solution and incubated in a water bath shaker at 55℃ for 1 hour (keeping the drug concentration of each group at 1mg / ml and the total phospholipid concentration of the system at 30mg / ml). Finally, liposomes with a total volume of 15ml were obtained. After removal, each group was placed in a labeled dialysis bag and dialyzed at 4℃ for 24h. (The dialysis membrane has a molecular weight cutoff of 3500 Da.)

[0076] (2) Preparation of polylysine-coated active factor liposomes (H-Lip-ε-PL): Dissolve ε-polylysine (ε-PL) at a concentration of 10 mg / ml in a flat-bottomed glass bottle, dispense into 4 ml / bottle, add magnetic stir bar to three groups of bottles and place on a common multi-channel magnetic stirrer and rotate at 1200 rpm. The blank liposomes (K-lip), the liposomes encapsulated with active short peptides (H-lip), and the active short peptide liposomes (Fitc-H-lip) prepared in (1) above are respectively drawn into 10 ml syringes. Each group of liposomes is added dropwise to the three groups of bottles above and stirred at a constant speed for 1 hour. After stirring, they are drawn out into clean EP tubes and centrifuged at 15000 rpm and 4℃ for 15 minutes in a centrifuge at 4℃. The supernatant of each group is discarded and resuspended to obtain the liposomes prepared in this invention.

[0077] (3) Preparation of thiolized hyaluronic acid / active factor liposomes / adipose stem cell composite hydrogel:

[0078] ① Modification of thiolized hyaluronic acid: Hyaluronic acid was added to distilled water and stirred to dissolve. A hyaluronic acid solution was obtained after the solid was completely dissolved. L-cysteine ​​hydrochloride was then added to the hyaluronic acid solution, followed by EDC / NHS and stirring to dissolve in the dark. The pH of the solution was adjusted to 4.7, and L-cysteine ​​hydrochloride was grafted to obtain a thiolized hyaluronic acid complex. Then, the complex was dialyzed for 3 days each with deionized water at pH 5, deionized water at pH 5 containing 1 wt% NaCl, and deionized water at pH 5. Finally, the complex was freeze-dried in the dark to obtain thiolized hyaluronic acid.

[0079] ② Preparation of composite hydrogel: It was used to dissolve a certain proportion of thiolized hyaluronic acid in the solution of active factor liposomes using a magnetic stirrer. After dissolution, a specific volume of α-ketoglutarate and N-hydroxysuccinimide was added as a gel precursor solution. 1M sodium hydroxide (NaOH) was gradually added dropwise to the precursor solution at a stirring speed of 1000 rpm to adjust the pH of the system to 7-8. The solution was then immediately removed, dispensed into templates, and placed in a 37℃ water bath. After the system gelled, it was sealed and stored in a 4℃ refrigerator for later use.

[0080] ③ Preparation of hydrogels loaded with active peptide liposomes containing adipose-derived stem cells (ADSCs / H-lip / ε-PL / HASH): Prepare a simple gel precursor solution containing active peptide liposomes (DDS) as described above. Filter the solution through a 0.22µm filter in a laminar flow hood. After sterilization by UV irradiation, gradually add sterile 1M sodium hydroxide (NaOH) dropwise to the precursor solution to adjust to neutrality. Incubate at 37°C for 10 minutes. Remove the solution and gently soak the gel system repeatedly with PBS containing 0.1% triple antibodies. Incubate at 37°C for 30 minutes. Remove the solution and purify it 5-7 times with stem cell-specific culture medium. Place the gel in a laminar flow hood for later use. Digest adipose-derived stem cells cultured to the third generation and adjust the cell count to 10^4 cells per milliliter. Add 1 ml of each adipose-derived stem cell suspension to the gel surface and incubate at 37°C for 1 day before use.

[0081] The preparation steps of the silk fibroin / polyhexamethylene biguanide electrospun membrane on the scaffold are as follows:

[0082] (1) Extraction of silk fibroin: Take the cut silkworm cocoons and add them to a 0.02M sodium carbonate aqueous solution, boil, degummed until no yellow gelatinous precipitate is produced; then wash the degummed silkworm cocoons with a large amount of deionized water, dry them after washing to obtain dry silk fibroin, dissolve the dry silk fibroin in a saturated 9.3M lithium bromide solution for 4 hours, dialyze and freeze dry to obtain silk fibroin.

[0083] (2) Preparation of silk fibroin / polyhexamethyl biguanide electrospinning solution: The obtained freeze-dried silk fibroin (SF) was dissolved in hexafluoroisopropanol (HFIP) to form a spinning solution. A certain mass of (PHMB) was weighed and dissolved in the silk fibroin spinning solution at a speed of 120 rpm. The mass-volume ratio of polyhexamethyl biguanide in the electrospinning solution was 0.3 wt% and 1 wt%, and they were named SF / PH0.3 and SF / PH1.

[0084] (3) Preparation of silk fibroin / polyhexamethyl biguanide electrospun membrane: Take the above-prepared and stirred spinning solution and transfer it to the syringe equipped with an 18G needle. Place it on the electrospinning machine, apply a high voltage of 23KV to the injection pump and equip it with a suitable push speed and high voltage electric field. Adjust the receiving distance to 10-12cm. Fix a layer of tin foil on the surface of the receiving shaft to receive the spinning. After spinning, peel off the sample along with the tin foil and place it in a constant temperature vacuum drying oven to dry and remove the unevaporated solvent. Store the sample for later use.

[0085] Synthesis of a double-layer biomimetic artificial skin scaffold:

[0086] The silk fibroin / polyhexamethylene biguanide electrospun membrane was combined with a thiolated hyaluronic acid / H active factor composite hydrogel during gel formation via Michael addition reaction. The silk fibroin / polyhexamethylene biguanide electrospun membrane served as the upper layer of a bilayer biomimetic artificial skin scaffold, while the thiolated hyaluronic acid / H active factor composite hydrogel served as the lower layer. Fibroblasts and adipose-derived stem cells were seeded in the upper and lower layers, respectively, and the bilayer scaffold was combined through gas-liquid co-culture. This bilayer scaffold can also be cultured in layers for wounds of different depths and sizes.

[0087] The specific implementation method is as follows:

[0088] Preparation method of antibacterial membrane on the upper layer of artificial skin:

[0089] (1) Preparation of spinning solution: Take the cut silkworm cocoons and add them to a 0.02M sodium carbonate aqueous solution. Boil and degumme until no yellow gelatinous precipitate is produced. Wash the degummed silkworm cocoons with a large amount of deionized water, and dry them to obtain dry silk fibroin. Dissolve the dry silk fibroin in a 9.3M lithium bromide solution for 4 hours, dialyze and freeze dry to obtain freeze-dried silk fibroin. Dissolve the freeze-dried silk fibroin in hexafluoroisopropanol (HFIP) to prepare a spinning solution. Weigh a certain mass of polyhexamethylene biguanide and dissolve it in the spinning solution at a speed of 120 rpm. The mass-to-volume ratio of polyhexamethylene biguanide in the electrospinning solution is 0.3-1 wt%.

[0090] (2) Preparation of antibacterial membrane: Preparation of silk fibroin / polyhexamethyl biguanide electrospun membrane: Take the spinning solution prepared in (1) into a syringe equipped with an 18G needle, place it on an electrospinning machine, and electrospin under the conditions of 23KV voltage and receiving distance of 10-12cm. After spinning, the sample is dried in a constant temperature vacuum drying oven to remove the unvolatile solvent, and the antibacterial material layer is obtained.

[0091] Performance characterization:

[0092] The antibacterial membrane has a main diameter distribution of about 1 μm, which allows for cell growth and possesses an excellent microstructure. (See...) Figure 2 Furthermore, this antibacterial membrane possesses excellent antibacterial and hemostatic properties, as well as good mechanical properties. It exhibits excellent hemostatic effects in rat models of massive skin hemorrhage and liver hemorrhage, making it suitable as an emergency treatment material for burns and acute trauma. In addition, this material demonstrates excellent tissue healing ability in full-thickness infected wounds, producing significant collagen deposition and promoting wound closure rate.

[0093] Preparation method of active factor liposomes

[0094] (1) Soybean phospholipids and cholesterol were dissolved in anhydrous ethanol at a mass ratio of 5:2. The solution was placed in a vacuum rotary evaporator and evaporated at 45°C and 200 rpm to form a uniform phospholipid film.

[0095] (2) Add a certain volume of citric acid solution with pH 4 to maintain the phospholipid concentration at 30 mg / ml, hydrate the above phospholipid membrane, and then place it in an ultrasonic bath to disperse it evenly. Use 300W, turn on for 30 seconds and turn off for 30 seconds, and sonicate each 6 ml of liposomes for 1 minute.

[0096] (3) The above liquid was transferred into a 100mM sodium phosphate solution and dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 3500 Da to obtain the required blank liposomes without inner encapsulation.

[0097] (4) Preparation of liposomes encapsulated with active peptides: Under the conditions of maintaining the concentration of active factor at 1 mg / ml and the concentration of phospholipid in the total system at 30 mg / ml, the active factor at a concentration of 1 mg / ml was dissolved in PBS and added to blank liposomes. After incubating in a water bath shaker at 55°C for 1 hour, the liposomes were dialyzed in a dialysis bag with a molecular weight cutoff of 3500 Da at 4°C for 24 hours to obtain encapsulated active liposomes.

[0098] (5) Preparation of polylysine-coated active factor liposomes: Prepare an ε-polylysine solution with a concentration of 10 mg / ml. Take 4 ml of the solution and add the encapsulated active peptide liposomes dropwise while stirring at 1200 rpm. Keep stirring for 1 hour. Remove the solution and centrifuge at 15000 rpm for 15 minutes at 4°C. Dissolve the solution in the appropriate solvent and store at 4°C to obtain the liposomes of this invention.

[0099] Performance characterization:

[0100] From the appendix Figure 3 It can be seen that the H-LIP-ε-PL liposome group has a superior pro-angiogenic effect, which can promote tubule formation and generate a large number of new capillaries within 24 hours. This active factor can promote angiogenesis and release the active factor slowly while maintaining the activity of growth factors.

[0101] Preparation method of artificial skin sublayer - bioactive hydrogel

[0102] (1) Preparation of modified hyaluronic acid:

[0103] 4g of hyaluronic acid was added to 1L of distilled water and stirred to dissolve, thus preparing a homogeneous solution. Then, 0.8g of L-cysteine ​​hydrochloride was added to the hyaluronic acid solution. Next, EDC / NHS was added and stirred to dissolve in the dark. The pH of the solution was adjusted to 4.7 to obtain the grafted modified thiolized hyaluronic acid complex. Then, the solution was dialyzed for 3 days each with deionized water at pH 5, 1wt% sodium chloride solution, and deionized water at pH 5, respectively. Finally, the solution was freeze-dried in the dark to obtain thiolized hyaluronic acid.

[0104] (2) Next, the preparation of the composite hydrogel: After sonicating an appropriate amount of liposome solution encapsulating active factors, 4% mercapto-modified hyaluronic acid was added to the stirrer. After dissolution, 0.02% α-ketoglutarate and 1% N-hydroxysuccinimide complex were added. Under a stirring speed of 1000 rpm, 1M sodium hydroxide solution was gradually added dropwise to adjust the pH of the system to 7-8. The system was then placed in a 37℃ water bath to form a gel. The gel was then sealed and placed in a 4℃ refrigerator for later use.

[0105] (3) Next, the preparation of liposome hydrogels containing active peptides loaded with stem cells: Take an appropriate amount of liposome solution encapsulating active factors and add 4% thiolated hyaluronic acid by mass through a stirrer. After dissolution, add 0.02% α-ketoglutarate and 1% N-hydroxysuccinimide complex. Place it in a clean bench, filter it with a 0.22 μm filter, sterilize it by UV irradiation, and gradually add sterile 1M sodium hydroxide solution. After adjusting the system to neutral, place it in a carbon dioxide incubator at 37°C for 10 minutes to stabilize the gel. After taking it out, add PBS solution containing 0.1% triple antibody to rinse the gel system, and then place it in a carbon dioxide constant temperature incubator at 37°C for 30 minutes. Take it out and add stem cell-specific culture medium to purify it 5-7 times. Take stem cells cultured to the third generation and adjust the cell number to 10. 4 1 ml of stem cell suspension was added to the surface of the gel and placed in a carbon dioxide incubator at 37°C for 24 hours to obtain a stem cell-loaded liposome hydrogel containing active peptides.

[0106] Performance characterization:

[0107] From the appendix Figure 2 The active factor liposomes with a pore size of about 100 μm and coated with ε-polylysine are uniformly distributed on the inner wall of the gel scaffold due to electrostatic interaction and slowly release the active factor.

[0108] From the appendix Figure 4Comparison of the groups revealed that the combined treatment group, based on HE, Masson, and CD31 fluorescence staining results, exhibited the strongest tissue healing ability, producing a large number of new blood vessels at the defect site, and showing the best collagen deposition, thus promoting wound closure. Therefore, the combined treatment group (ADSCs / CH02-lip / ε-PL / HASH) demonstrated superior therapeutic efficacy compared to other experimental groups. This bioactive hydrogel is characterized by low immunogenicity and high biocompatibility.

[0109] Method for preparing a double-layer artificial skin scaffold:

[0110] (1) After the antibacterial membrane is fully dried, it is moistened and incubated at 37°C for one hour with the composite hydrogel precursor solution. The prepared composite hydrogel is placed on the surface of the moistened antibacterial membrane. The entire material is immersed in a petri dish containing the precursor solution. After adjusting the precursor solution to neutral with 1M sodium hydroxide, the petri dish is placed at 37°C for one hour to form a double-layer artificial skin scaffold-antibacterial membrane / bioactive hydrogel.

[0111] (2) The scaffold can be used to cultivate biomimetic dermal and epidermal cell scaffolds on a large scale in a layered culture environment such as gas-liquid co-culture. The active factors used in the cell culture environment are all derived from mild active factors extracted by the patient and platelet lysates. The dermal cells are derived from a co-culture of the patient's own adipose stem cells and endothelial cells, and have the characteristics of low immunogenicity and high biocompatibility.

[0112] Performance characterization:

[0113] After freeze-drying, scanning electron microscopy revealed that the upper scaffold consisted of electrospun nanofibers with a diameter of approximately 1 μm, while the lower hydrogel layer contained ε-polylysine-coated liposomes with a pore size of approximately 100 μm, which, due to electrostatic interactions, were uniformly distributed on the inner wall of the gel scaffold, slowly releasing active factors. Crosslinking occurred at the interface between the two layers. Figure 2 This dual-layer material can be customized according to the patient's specific condition to achieve personalized treatment, and it is expected to become a highly promising artificial skin scaffold material.

[0114] The preparation method of this invention is mild, natural and non-toxic, which is conducive to cell growth. It can also rapidly construct micro-tissues in vitro, and has degradable, antibacterial and anti-tensile effects. In addition, it has good strength, hemostatic, antibacterial and biocompatibility properties.

[0115] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A method for preparing a composite stem cell active biomaterial, characterized by, The composite material comprises a double-layer structure, wherein the lower layer is an active material layer in the form of a hydrogel composed of active liposomes and stem cells, and the upper layer is an antibacterial layer; The preparation steps of the active liposomes are as follows: 1) Dissolve soybean phospholipid and cholesterol in anhydrous ethanol according to a mass ratio of 5:2, place the dissolved substance in a vacuum rotary evaporator, spin dry at a temperature of 45 DEG C and a rotation speed of 200 rpm, and form a uniform phospholipid film; 2) Add a citric acid solution with a pH of 4 to keep the phospholipid concentration at 30 mg / mL, hydrate the phospholipid film, and then place it in an ultrasonic wave for uniform dispersion, 300 W, 30 s on and 30 s off, 1 minute of ultrasonic wave for every 6 mL of liposomes; 3) Transfer the liquid into a sodium phosphate solution with a concentration of 100 mM, and dialyze for 24 hours with a dialysis bag with a molecular weight cut-off of 3500 Da, so as to obtain the required empty liposomes without an encapsulated substance; 4) Preparation of liposomes encapsulating active factors: keep the active factor concentration at 1 mg / mL and the phospholipid concentration in the total system at 30 mg / mL, dissolve the active factor with a concentration of 1 mg / mL in PBS and add it into the empty liposomes, incubate in a water bath shaker at 55 DEG C for 1 hour, and then dialyze in a dialysis bag with a molecular weight cut-off of 3500 Da at a temperature of 4 DEG C for 24 hours, so as to obtain the liposomes encapsulating active factors; 5) Preparation of active factor liposomes coated with polylysine: prepare an epsilon-polylysine solution with a concentration of 10 mg / mL, take 4 mL, and add the liposomes encapsulating active factors drop by drop under the condition of rotation stirring at a rotation speed of 1200 rpm, keep stirring at the rotation speed for 1 hour, take out, centrifuge at a rotation speed of 15000 rpm for 15 minutes in a centrifuge at 4 DEG C, and then store after being dissolved in a corresponding solvent at 4 DEG C, so as to obtain the active liposomes; The preparation steps of the active material layer are as follows: 1) First, preparation of thiolated hyaluronic acid: take 4 g of hyaluronic acid and add it into 1 L of distilled water, stir and dissolve to prepare a uniform solution; then add 0.8 g of L-cysteine hydrochloride into the hyaluronic acid solution; then add EDC / NHS under light shielding and stirring, adjust the solution pH to 4.7, so as to obtain the grafted and modified thiolated hyaluronic acid complex; then dialyze with deionized water with a pH of 5, a sodium chloride solution with a concentration of 1 wt%, and deionized water with a pH of 5, respectively, for 3 days, and then freeze-dry under light shielding to obtain the thiolated hyaluronic acid; 2) Second, preparation of composite hydrogel: take an appropriate amount of active liposome solution after ultrasonic treatment, add 4% thiolated hyaluronic acid into the stirrer, dissolve completely, and then add 0.02% alpha-ketoglutaric acid and 1% N-hydroxysuccinimide complex; gradually add 1 M sodium hydroxide solution drop by drop under the condition of stirring at a rotation speed of 1000 rpm, adjust the system pH to 7-8, take out, and form a gel in a water bath at 37 DEG C, and then seal the gel and store in a refrigerator at 4 DEG C; 3) Again is the preparation of active liposome hydrogel loaded stem cells: take the right amount of active liposome solution by stirrer into the mass fraction of 4% sulfhydryl hyaluronic acid, dissolved, then add the mass fraction of 0.02% alpha-ketoglutaric acid and 1% N-hydroxysuccinimide compound; it is placed in the clean bench, with 0.22um filter head filtration, after ultraviolet irradiation sterilization, gradually added by sterile 1M sodium hydroxide solution, adjust the system to neutral, placed in the temperature of 37℃ carbon dioxide incubator for 10 minutes, stable into gel; take out and add 0.1% PBS solution containing triantigenic antibody to rinse the gel system, and then placed in the temperature of 37℃ carbon dioxide incubator for 30 minutes; take out and add stem cell special culture medium to purify it for 5-7 times; take the third generation of stem cells, adjust the cell number to 10 4 individuals per milliliter, take 1mL stem cell suspension, add to the surface of the gel, and place in the temperature of 37℃ carbon dioxide incubator for 24 hours, to obtain the active liposome hydrogel loaded stem cells; The antibacterial layer is composed of antibacterial material polyhexamethylene biguanide and silk fibroin by electrospinning, and the preparation steps of the antibacterial layer are as follows: 1) Extraction of silk fibroin: Take the cut cocoon and add it to a 0.02M sodium carbonate solution, boil, degum until no yellow gum precipitate is produced; wash the degummed cocoon with a large amount of deionized water, dry after washing to obtain dry silk fibroin, take the dry silk fibroin and dissolve it in a 9.3M lithium bromide solution for 4 hours, dialysis and freeze-drying to obtain freeze-dried silk fibroin; 2) Preparation of silk fibroin / polyhexamethyl biguanide electrospinning solution: Dissolve the freeze-dried silk fibroin in hexafluoroisopropanol (HFIP) as a spinning solution, weigh a certain amount of polyhexamethyl biguanide and dissolve it in the spinning solution at a speed of 120 rpm, the mass / volume ratio of polyhexamethyl biguanide in the electrospinning solution is 0.3-1wt%; 3) Preparation of silk fibroin / polyhexamethyl biguanide electrospinning membrane: Take the above spinning solution in a syringe with an 18G needle, place it on the electrospinning machine, electrospin under the conditions of a voltage of 23KV and a receiving distance of 10-12cm, dry the spun sample in a constant temperature vacuum drying oven to remove the non-volatile solvent, and obtain an antibacterial material layer.

2. The method of claim 1, wherein the active biomaterial is a composite stem cell. 2 The active factor is derived from marine biological materials, or terrestrial biological materials, or short peptide active substances derived from microorganisms manufactured by genetic engineering technology, with a molecular weight of 2KD to 100kD.

3. The method of claim 1, wherein the active biomaterial is a composite stem cell.

3. The method of claim 1, wherein the active biomaterial is a composite stem cell. The preparation method of the double-layer composite material is: using Michael addition reaction technology, the electrospinning antibacterial material layer containing silk fibroin / polyhexamethyl biguanide is compounded with active liposome hydrogel containing thiolated hyaluronic acid during gel formation, the electrospinning membrane is the upper layer of the double-layer composite material, and the active liposome hydrogel is the lower layer, further planting fibroblasts and adipose stem cells in the upper and lower layers, and obtaining the active biomaterial of composite stem cells by gas-liquid co-culture.

4. The method of claim 1, wherein the active biomaterial is a composite stem cell. 5 The stem cells are one of adipose stem cells, umbilical cord stem cells, and bone marrow mesenchymal stem cells, or from the patient's own body, or from terrestrial animals.

5. The use of the active biomaterial of the composite stem cells according to any one of claims 1-4 in artificial skin, characterized in that, The active biomaterial of composite stem cells is applied to artificial skin.

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