CN116785500A - A kind of degradable polymer tissue engineering scaffold and its preparation method and application - Google Patents

Abstract

The invention discloses a preparation method of a degradable polymer tissue engineering scaffold, which comprises the following steps: taking a hydrogel dressing based on chitin derivatives with the thickness of 2-8 mm as an active polymer layer, wherein the porosity in the active polymer layer is 1-90%, and the pore size is 100-4000 mu m; preparing an absorbent layer on the active polymer layer, wherein the absorbent layer is selected from at least one of hydrophilic fiber layer, alginate layer, semipermeable polyurethane film and hydrocolloid layer to obtain active polymer layer-absorbent layer; and (3) sequentially and uniformly spraying a mixed solution of human serum albumin and silk fibroin, thrombin and a fibrin solution on the surface of the active polymer layer-absorbing layer to form a mesenchymal stem cell attaching layer, so as to obtain the degradable polymer tissue engineering scaffold. The special skin stent system produced by the invention has extremely high biocompatibility, has no rejection reaction after transplantation, and can fully ensure the use safety of the composite skin stent.

Description

A degradable polymer tissue engineering scaffold and its preparation method and application

Technical field

The invention belongs to the field of tissue regeneration medicine, and in particular relates to a degradable polymer tissue engineering scaffold and its preparation method and application.

Background technique

Chitin derivatives (chitin polyesters) are obtained by chitin esterification in the presence of aliphatic and cyclic acid anhydrides and certain hydroxy acids. Chitin, from which calcium carbonate has been previously removed, is acylated in the presence of the selected catalyst (which also serves as the reaction medium) and the selected acid anhydride or hydroxy acid. Chitin can be esterified at one or two positions. Chitin substitutes can be derivatives of hydroxy acids (lactic acid, glycolic acid), linear saturated aliphatic anhydrides (acidic residues with chain lengths from C2 to C8, e.g. from acetic anhydride to octenoic anhydride and from e.g. acetic acid acrylic anhydride , anhydride of butylene acrylic anhydride), branched aliphatic unsaturated anhydride (methacrylic anhydride, 2-butenoic anhydride) or cyclic acid anhydride (maleic anhydride, glutaric anhydride, fluoric anhydride, phthalic anhydride ). Chitin can be substituted at one or two positions using one or two substituents of the same or different chain lengths.

In the case of leaching, "chitin derivatives" are present in catalysts and linear saturated aliphatic anhydrides with acidic residual chains from C2 to C8 (e.g., from acetic anhydride to octene anhydride and from, for example, acetic anhydride to propionic anhydride, The presence of butyric-propionic anhydride), branched unsaturated fatty acid anhydrides (e.g., methacrylic anhydride, 2-butenoic anhydride), or cyclic anhydrides (glutaric anhydride, fluoric anhydride, phthalic anhydride) Below, chitin polyester produced in chitin esterification reaction.

Chitin and chitosan act as chemoattractants for macrophages or neutrophils, thereby initiating the healing process and stimulating granulation and re-epithelialization processes while limiting scarring. In addition, chitin and its derivatives themselves possess antibacterial and antifungal properties. It is speculated that "the cationic group combined with the anion of the bacterial cell wall can inhibit biosynthesis", and that chitin will disrupt the molecular transport of the bacterial cell wall and accelerate its death.

The application of chitin and its derivatives in regenerative medicine has the following advantages:

(1) High biocompatibility, can induce cell communication and tissue regeneration;

(2) Has relatively suitable mechanical and physical properties;

(3) Biodegradability: Gradually degrades at a rate that does not exceed the appropriate rate for regeneration and remodeling of damaged wounds, and does not induce an immune response; its degradation products are non-toxic. This process is based on the enzymatic hydrolysis reaction of acetyl residues (mainly lysozyme in vivo);

(4) Can be used for adhesion, proliferation, migration, and differentiation of stem cells and other cells (polymers can be combined with growth factors, viscous proteins, serum proteins, fibrin, and thrombin to promote cell adhesion and be used for wound hemostasis) ), and promote stem cell migration during culture;

(5) Chitin and its derivatives have certain physical antibacterial abilities and can also be combined with antibiotics and chemical antibacterial agents to avoid wound infection, which is of great significance for the healing of difficult-to-heal wounds;

(6) Chitin and its derivative polysaccharides can be used as drug carriers. Their slow dissolution by body fluids can ensure sustained and sustained release of drugs.

At present, skin grafting is still the main treatment for severe burns, and autologous skin is often used for transplantation. However, when the wound area is large, the supply of autologous skin is limited, which cannot meet medical needs. Moreover, autologous skin transplantation must rely on surgery. , which will increase the patient's pain during treatment and is not conducive to the patient's wound healing. In addition, although skin replacement products have appeared on the market and are used clinically, they induce strong immune responses due to their poor biocompatibility, or the presence of raw materials Animals or human perinatal waste tissues that are difficult to trace have certain safety risks and cannot be produced on a large scale, seriously affecting the market promotion process of similar products.

Contents of the invention

The primary purpose of the present invention is to provide a degradable polymer tissue engineering scaffold.

Another object of the present invention is to provide a method for preparing the above-mentioned degradable polymer tissue engineering scaffold.

Another object of the present invention is to provide the application of the above-mentioned degradable polymer tissue engineering scaffold.

The present invention is implemented as follows: a method for preparing a degradable polymer tissue engineering scaffold, which method includes the following steps:

(1) A hydrogel dressing based on chitin derivatives with a thickness of 2 to 8 mm is used as the active polymer layer, wherein the porosity in the active polymer layer is 1 to 90% and the pore size is 100 to 4000 μm;

(2) Prepare an absorbent layer on the active polymer layer. The absorbent layer is selected from at least one of a hydrophilic fiber layer, an alginate layer, a semi-permeable polyurethane membrane, and a hydrocolloid layer to obtain an active polymer layer. -absorbent layer;

(3) Spray human serum albumin, silk fibroin, thrombin and fibrin solutions evenly on the surface of the active polymer layer-absorbent layer in sequence to form a mesenchymal stem cell attachment layer to obtain a degradable polymer tissue engineering scaffold.

Preferably, the chitin derivative is selected from any one of hydroxy acids, linear saturated aliphatic acid anhydrides, branched chain aliphatic unsaturated acid anhydrides and cyclic acid anhydrides;

The mixture of chitin derivatives is a combination of two or more chitin derivatives, or a chitin derivative and other biocompatible, biodegradable polymers, and the polymer is selected from polylactide , at least one of polyethylene glycol and 3-hydroxybutyric acid polyester.

Preferably, the hydroxy acid is lactic acid or glycolic acid; the acid group chain length of the linear saturated aliphatic anhydride is C2 to C8, and is selected from acetic anhydride to octenoic anhydride, acetic acid-propionic anhydride, butyric acid-propionic anhydride. Any one of the acid anhydrides; the branched aliphatic unsaturated acid anhydride is methacrylic anhydride or 2-butenoic anhydride; the cyclic acid anhydride is selected from maleic anhydride, glutaric anhydride, acetic anhydride and ophthalmic anhydride. Any of phthalic anhydride.

Preferably, in step (3), the spraying process includes the following steps: adding a mixed solution containing human serum albumin and silk fibroin at a concentration of 1ug/mL to 100mg/mL, and adding 0.1 to 100mg/mL of silk fibroin. Spray 2 mL/s evenly on the surface of the polymer layer-absorbent layer for 5 to 60 seconds. After the layer material is basically dry, spray a solution containing 0.1 to 000 U/mL human thrombin at a flow rate of 0.1 to 2 mL/s for 5 to 60 seconds. After an interval of 5 minutes to 5 hours, spray a solution containing 0.2 to 5.0 mg/mL fibrinogen at a constant flow rate of 0.1 to 2 mL/s for 5 to 60 seconds, let it stand at 37°C for 5 minutes to 5 hours, and dry.

The invention further discloses the degradable polymer tissue engineering scaffold obtained by the above preparation method.

The invention further discloses the application of the above-mentioned degradable polymer tissue engineering scaffold in preparing drugs or drug carriers for regeneration and remodeling of damaged wounds.

The invention further discloses a composite tissue engineering scaffold system, which system includes the above-mentioned degradable polymer tissue engineering scaffold and stem cells loaded and combined on the degradable polymer tissue engineering scaffold.

The invention further discloses the application of the above-mentioned composite tissue engineering scaffold system in preparing wound healing drugs and/or skin and tissue regeneration drugs.

The present invention overcomes the shortcomings of the existing technology and provides a degradable polymer tissue engineering scaffold and its preparation method and application. The present invention uses a hydrogel dressing based on chitin derivatives with a thickness of 2 to 8 mm as an active polymer layer, wherein the porosity in the active polymer layer is 1 to 90% and the pore size is 100 to 4000 μm; secondly , prepare an absorption layer on the active polymer layer, the absorption layer is selected from at least one of a hydrophilic fiber layer, an alginate layer, a semi-permeable polyurethane membrane, and a hydrocolloid layer, to obtain an active polymer layer-absorption layer layer; finally, human serum albumin, silk fibroin, thrombin and fibrin solutions are evenly sprayed on the surface of the active polymer layer-absorbent layer in sequence to form a mesenchymal stem cell adhesion layer, and a degradable polymer tissue engineering scaffold is obtained. .

The active polymer layer is composed of a chitin derivative (0.1 to 100% by volume) or a mixture of chitin derivatives (two or more chitin derivatives or in combination with other commonly available biocompatible, bioavailable Biocomposite materials based on one or more chitin derivatives made from biodegradable polymers, such as polylactide, polyethylene glycol, 3-hydroxybutyric acid polyester and other chitin derivatives. Chitin derivatives refer to compounds with hydroxy acids (such as lactic acid, glycolic acid), linear saturated aliphatic anhydrides (acid group chain length C2 ~ C8), (for example, from acetic anhydride to octenoic anhydride and from such as acetic acid propionic anhydride , butyric acid-propionic anhydride), branched aliphatic unsaturated anhydride (such as methacrylic anhydride, 2-butenoic anhydride) or cyclic acid anhydride (maleic anhydride, glutaric anhydride, fluoric anhydride, phthalic anhydride dicarboxylic anhydride). Chitin derivatives can undergo substitution reactions with one or two substituents of different lengths via one or two of their sites. The active polymer layer has a three-dimensional structure (including pores (straight holes or coiled holes) with a diameter of 0.1 mm to 4.0 mm, depending on the type of wound, or forming holes of different sizes, and the proportion of holes in the dressing material is 1 to 90 volume%). The active polymer layer can be further combined with an absorbent layer that aids in wound repair.

In addition, the dressing properties of the active polymer layer can also be directly made into a hydrogel with suspended chitin derivatives or their mixture molecules. The hydrogel stores a large amount of water (5-85% by volume) and can be directly applied to the The wound surface, due to its hydrophilic properties, can ensure concentrated moisturizing and hydration of dry wounds, and only absorbs and binds wound exudate in a small area, so it can be used for wounds without large-area exudates. This type of active polymer layer dressing can be used for second-degree burns and abrasions over areas where skin has been removed during skin grafting.

In the absorption layer, at least one of a hydrophilic fiber layer, an alginate layer, a semi-permeable polyurethane membrane, and a hydrocolloid layer can be selected according to functional requirements.

(1) Hydrophilic fiber layer

Based on carboxymethyl cellulose (or other cellulose derivatives with similar physical and chemical properties), the cellulose derivative content ranges from 5 to 100% by volume, the layer thickness ranges from 0.1mm to 5.0mm, and the shape and size depend on the inner Layers, in the form of hydrophilic fibers, are compressed into sheets. The hydrophilic fibrous layer contacts the wound exudate to form a gel coating, in which the exudate is absorbed and trapped in the fibrous structure of the hydrophilic fibrous layer, thus eliminating pathogenic bacteria. The hydrophilic fiber layer is preferred for wounds with a high risk of infection that have a large amount of exudate. In addition, silver ions (0.1 to 2 volume %) can be added to provide the hydrophilic fiber layer with an antibacterial function.

(2)Alginate layer

Based on alginate layer - calcium and sodium-calcium alginates obtained from sodium-calcium derivatives of alginic acid (D-mannuronic acid and L-glucuronic acid) and polymers obtained from marine brown algae (GG, MM , MG blocks; L-glucuronic acid 25 to 85 volume %, mannuronic acid 20 to 70 volume %, the percentage content of glucuronic acid exceeds the percentage content of mannuronic acid; calcium ion content 0.1 to 10.0 volume %, sodium ion Content 0.1~2.0%). The alginate layer is prepared from fibers in a compressed form. As wound exudate is absorbed, a gel incorporating the exudate forms around the fibers. The phenomenon of gelation is based on the exchange of calcium ions on the fiber surface and sodium ions in the exudate. The alginate layer can be used for moderate and severe exudative wounds.

(3) Semi-permeable polyurethane membrane

The polyurethane content is 15~100% by volume and the layer thickness is 0.1mm~5.0mm, forming a barrier (waterproof) between microorganisms and water, and allowing gas to evaporate from the wound surface, while ensuring a moist internal wound environment and a dry surface, and is easy to Remove from layers that are in direct contact with the wound. Therefore, semi-permeable polyurethane membranes are suitable for mild and moderate exuding wounds.

(4) Hydrocolloid layer

The hydrocolloid layer is in the form of a plate, including an outer protective layer and an inner layer. The outer protective layer is semi-permeable (purpose: to protect against contamination and invasion by pathogenic microorganisms), the inner layer contains carboxymethylcellulose (sodium carboxymethylcellulose and other types) suspended in hydrophobic pectin A mixture of hydrophilic molecules (ensure suitable humidity) and colloids (ensure a slightly acidic pH) of carboxymethyl cellulose. During contact with wound exudate, the hydrocolloid layer swells and becomes a gel due to the interaction of enlarging molecules. Condensation into a homogeneous viscous colloid system in which the free spaces are filled with absorbed exudate. This hydrocolloid layer maintains a moist wound environment, constant temperature and slightly acidic pH conditions (reduces pain, acidic pH reduces prostaglandin production of PGE2 that sensitizes nerve endings). The hydrocolloid layer can be used in mild to moderate exuding wounds.

The invention uses degradable polymer combined with stem cell technology to have a combined therapeutic effect and the ability to repair difficult-to-heal wounds such as deep third-degree burns. Because chitin is cationic, and it primarily mediates electrostatic binding to anionic glycosaminoglycans (GAGs), proteoglycans, and other negatively charged molecules. Mesenchymal stem cells can secrete a large amount of cytokines/growth factors, which can directly bind to GAG and be highly enriched and repair the material surface, thereby assisting stem cells in promoting orderly regeneration of surrounding tissues.

The present invention has an intrinsic antibacterial effect, while allowing the binding and controlled release of exogenous antibacterial factors, and the interaction between the antibacterial effect of chitin and the tissue anti-inflammatory effect of MSC has dual effects in anti-infection and promoting tissue healing. The effect is significant compared to similar competing products (polymer mesh).

Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects:

(1) The present invention uses three spraying processes to evenly spray controllable doses of a mixed solution of human serum albumin and silk fibroin, thrombin and fibrin solutions on the surface of the absorption layer of the degradable polymer tissue engineering scaffold. On the one hand, this protective layer helps stem cells adhere and proliferate. At the same time, when the skin scaffold is attached to a wound, the human serum albumin on the skin scaffold can achieve wound protection (prevent tissue fluid from leaking out), thereby reducing the occurrence of wound ulcers. At the same time, it can also provide nutrients for damaged tissue and promote wound healing. When the skin stent is attached to the wound, it can also act on the damaged area through the interaction of the residual thrombin activity on it with the fibrin source in the patient's blood. To stop bleeding. Silk fibroin can increase the flexibility of the scaffold to a certain extent.

(2) In the present invention, after soaking the independently preserved composite scaffold material in the stem cell culture solution before use, stem cells from different sources can be attached to it to achieve better burn treatment effects and solve the dependence of patients with deep third-degree and large-area burns. The "painful situation" of repeated skin grafting can greatly reduce the difficulty of surgery and treatment costs for patients, and at the same time, it can significantly improve the healing effect of deep third-degree burn tissue. Due to the existence of stem cells, specific epidermis, dermis, blood vessels and other connective tissues can be locally induced to differentiate in an orderly manner, forming smaller scars. At the same time, it can promote the regeneration of special skin structures such as hair follicles and sweat glands; ultimately forming regenerated skin with physiological functions.

(3) In the present invention, since the skin stent attached to the wound is active, the skin stent can be integrated with the human skin after being attached to the affected part of the wound for a period of time, enabling permanent repair of the wound and avoiding the traditional repair process of multiple skin grafts. , the composite skin scaffold can reduce the pain suffered by patients due to repeated skin grafting.

(4) The materials used in the present invention come from a wide range of sources, including common inorganic and organic compounds. They are low-cost, convenient for large-scale production, and effectively reduce the contradiction between supply and demand of skin. In addition, this kind of composite skin scaffold is not only convenient to obtain materials, but also has a simple manufacturing process. It has the advantages of convenience for medical staff to operate, storage, transportation, commercial preparation and clinical application.

(5) The special skin scaffold system produced by the present invention has extremely high biocompatibility (the attached MSCs are also low immunogenic cells), and there is no rejection reaction after transplantation, which can fully ensure the safety of the composite skin scaffold. .

(6) Experiments on deep third-degree burns in large animals prove that the present invention combined with stem cells can promote the effective regeneration of advanced skin structures such as hair follicles.

(7) The addition of silk protein in the present invention can improve the flexibility of the polymer material to a certain extent.

Description of the drawings

Figure 1 is a diagram showing the effect of the degradable tissue engineering dressing (not combined with MSC) of the present invention on promoting wound healing; A: the dressing is applied to the wound, B: the dressing is highly integrated with the wound;

Figure 2 shows the combination process of stem cells and scaffolds; Figures 2A-B show the activation of the scaffold, before cells are seeded; Figures 2C-D show the state after cells are seeded;

Figure 3 shows the degradable tissue engineering scaffold combined with MSC synergy to improve the treatment effect of deep third-degree burns; among them. Left picture: Evaluation of the efficacy of a degradable tissue engineering scaffold without MSC on deep third-degree burn model pigs (relatively less new hair); Right picture: Evaluation of the efficacy of a degradable tissue engineering scaffold combined with MSC on deep third-degree burn model pigs ( Can induce hair regeneration).

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention and are not intended to limit the present invention.

Example 1

The preparation of the active polymer layer in this embodiment is the same as the content and connotation described in Example 1 of the reference patent CN 104582745 A, and specifically includes the following steps:

The active polymer layer is produced from a chitin derivative obtained by esterification with propionic anhydride (the concentration percentage of the chitin derivative in the dressing is 100%). Pour dissolved in acetone through a nozzle with a diameter of 2 to 8 mm at a rate of 1 to 3 l/min on a flat, non-absorbent surface (in the form of a belt) with a constant transport rate of 0.5 to 1.0 m/min and a width of 100 mm. Derivative chitin at a concentration of 10 to 30%. Once the polymer tape has cured, prepared ink pads (needle sizes from 0.098 to 3.98 mm) can be used to form holes in different routes, sizes and placement densities and shapes, thereby obtaining shapes depending on the type and purpose of the dressing. Polymer tape.

As a result of this process, an active polymer layer is produced, which has a three-dimensional porous structure or a non-porous structure (in the case of a porous structure, the diameter of the pores is 0.1 mm to 4.0 mm, straight or coiled, or mixed sized pores , the percentage of holes in a single dressing from 1 to 90%).

The prepared active polymer is immersed in a solution of the excipient and then is additionally impregnated with the excipient or other bactericidal substance by evaporation of the solvent and absorption from the solution. The active polymer layer is impregnated with excipients, such as 0.01% of Ag ⁺ ions and 0.01% of Ca ²⁺ ions. Active polymers, etc. are also impregnated with neomycin, bactericides and antibiotics with bacteriostatic properties.

The technical parameters characterizing a single-layer dressing depend on the active polymer layer thickness and porosity:

—Oxygen permeability (cm ³ /m ² ): 1000～9000;

—Elongation: 0.5~400%;

—Tensile strength (kg/cm ² ): 1 to 200;

—PBS absorption rate (0.2～50g PBS/24h/100cm ² dressing).

Example 2

The preparation of the active polymer layer in this embodiment is the same as the content and connotation described in Example 2 of the reference patent CN 104582745 A, and specifically includes the following steps:

The active polymer layer is produced by mixing three chitin derivatives (acetic acid derivative 10%, valeric acid derivative 10% and butyric acid derivative 80%). Pour ethanol added at a rate of 0.5 to 3 l/min through a nozzle with a diameter of 2 to 8 mm on a flat, non-absorbent surface (in the form of a belt) with a constant transport rate of 0.5 to 1.0 m/min and a width of 100 mm. It is a polymer mixture dissolved in 5 to 30% of excipients (described below). Next, after the poured layer solidifies, a living polymer is formed. The resulting active polymer is formed into a shape that depends on the type and purpose of the dressing. The active polymer layer is impregnated with excipients such as K ⁺ ions, 1.5% citric acid, and 0.001% bismuth salt. The active polymer is also impregnated with the bactericide: vancomycin (0.1%).

The technical parameters characterizing a single-layer dressing depend on the active polymer layer thickness and porosity:

—Oxygen permeability (cm ³ /m ² ): 500～3000;

—Elongation: 0.5~500%;

—Tensile strength (kg/cm ² ): 1 to 200;

—PBS absorption rate (0.2～50g PBS/24h/100cm ² dressing).

Example 3

The preparation process of the active polymer layer-absorbent layer in this embodiment includes the following specific steps:

The active polymer layer consists of a mixture of three chitin derivatives (obtained by the esterification of chitin with the following anhydrides in sequence: propane acetate 20%, butane propane 40% and methacrylic acid 20%), and the combinations are widely available Made of biocompatible biodegradable polymer polyethylene glycol (polyethylene glycol 20%). The active polymer layer was prepared as described in Example 1 and impregnated with excipients. The active polymer layer 1 is impregnated with 2.5% (mass, the same below) of Ag ⁺ ions, 0.1% of K ⁺ ions, and 0.1% of bismuth salt. Active polymer 1 was also impregnated with metronidazole (0.3%).

The absorbent layer is bonded to the active polymer layer 1 via a peripheral adhesive system (polyethylene adhesive), resulting in an active polymer layer-absorbent layer. Among them, the absorbent layer is made of commercially available hydrophilic fibers.

Example 4

The preparation process of the active polymer layer-absorbent layer in this embodiment includes the following specific steps:

The active polymer layer consists of a mixture of three chitin derivatives (obtained by the esterification of chitin with the following anhydrides in sequence: propane acetate 20%, succinate ester 40% and phthalate ester 20%), and Made by combining the widely available biocompatible biodegradable polymer polyethylene glycol (polyethylene glycol 20%). The active polymer layer was prepared as described in Example 1 and impregnated with excipients. The active polymer layer is permeated with 2.5% Ag ⁺ ions, 1.0% Ca ²⁺ ions, and 0.1% Zn ²⁺ ions. Active polymer 1 was also impregnated with metronidazole (0.3%).

The absorbent layer is bonded to the active polymer layer via Peripheral Adhesion System 2 (polyethylene adhesive). The absorbent layer is made of alginate.

Example 5

The preparation of the degradable polymer tissue engineering scaffold in this embodiment includes the following steps:

A mixed solution containing human serum albumin and silk protein at a concentration of 1ug/mL~100mg/mL and 0.1~100mg/mL silk protein is evenly sprayed on the surface of the active polymer layer-absorbent layer prepared in Example 3. The spraying process needs to be Carry out at a constant flow rate of 0.1 to 2mL/s. After the layer of material is basically dry, spray a solution containing 0.1 to 1000U/mL human thrombin at a constant flow rate of 0.1 to 2mL/s again. After an interval of 5min to 5h, spray the solution containing 0.1 to 2mL again at a constant flow rate of 0.1 to 2mL/s. Spray a solution containing 0.2 to 5.0 mg/mL fibrinogen at a constant flow rate /s, leave it at 37°C for 5 minutes to 5 hours, and dry to obtain a degradable polymer tissue engineering scaffold 1.

Example 6

24 hours before transplantation to the patient, prepare the composite tissue engineering scaffold system. The preparation process specifically includes the following steps:

(1) Pretreatment of degradable polymer tissue engineering scaffolds

Cut the degradable polymer tissue engineering scaffold prepared in the above Example 5 according to the required size and place it in a cell culture dish of different specifications (the absorbent layer after spraying with the stem cell attachment solution faces upward), add an appropriate amount of commercialized (containing gentamicin) (Cat No.: LF-BIO, MSCM Cat No.: LF0101A, add 2-5% serum substitute) and soak for 5 minutes-5 hours.

(2) Mesenchymal stem cell (MSC) culture

Umbilical cord mesenchymal stem cells isolated or commercially purchased from the original generation (the patient's autologous bone marrow, adipose-derived MSC or iPS cells derived from somatic cell transdifferentiation can also be used) are cultured to the logarithmic growth phase, and then digested with trypsin. Prepare a single cell suspension, centrifuge at 1000 rpm/min, resuspend in 1 mL of commercial professional culture medium (Cat No.: LF-BIO, MSCM Cat No: LF0101A plus 2-5% serum substitute), and let stand at 37°C for later use.

(3) Preparation of composite tissue engineering scaffold system (stem cell attached tissue engineering scaffold)

The resuspended MSCs were evenly planted on the surface of the stem cell adhesion layer of the pretreated tissue engineering scaffold (try to avoid cells falling into other areas of the culture dish), and then placed in a cell culture incubator at 37°C and 5 volume% _CO2 . Leave to incubate for 24 hours.

Application Example 1

The degradable polymer tissue engineering scaffold obtained in Example 3 of the present invention was attached to the patient's large-area burn surface. After 3 days, the inflammatory response of the wound was controlled, the surface was highly integrated with the tissue, and local tissue regeneration occurred, as shown in Figure 1.

Visible light and laser confocal microscopy of the degradable polymer tissue engineering scaffold obtained in Example 3 of the present invention confirmed that mesenchymal stem cells (MSC) can adhere to the surface of the scaffold and grow, as shown in Figure 2.

Application Example 2

The composite tissue engineering scaffold system prepared in Example 6 was applied to a large animal deep third-degree burn model (pig). The results are shown in Figure 3. It can be seen that the system can effectively promote tissue wound healing and induce hair follicles, etc. The skin appendage structure is regenerated, and the therapeutic effect is better than that of simple scaffold products without stem cells.

Application Example 3

The composite tissue engineering scaffold system cultured aseptically in Example 6 was carefully taken out with tweezers in a sterile environment in the operating room, with the absorption layer containing stem cells as the bottom surface, and evenly attached to the wound site after debridement. The composite The tissue engineering scaffold system can replace skin transplantation and meet the treatment needs of patients with deep third-degree and large-area burns.

It should be noted that charring of connective tissue often occurs in burn wounds of deep third degree and above. For such patients, the charred tissue and surrounding tissue need to be completely removed, fresh tissue is fully exposed, and after appropriate hemostasis treatment, stem cell tissue engineering The stent is attached to the treated wound surface and is combined with a hemostatic sponge and Vaseline gauze to bandage the wound in a timely manner. However, excessive adhesion between the dressing and the wound surface needs to be avoided. During the wound healing process, regularly replace gauze and other dressings and check the wound healing status in time. If the stent is absorbed too quickly, you can consider applying it again. However, under normal circumstances, the same wound can be healed within three times of application.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (8)

1. A method for preparing a degradable polymer tissue engineering scaffold, characterized in that the method includes the following steps: (1) A hydrogel dressing based on chitin derivatives with a thickness of 2 to 8 mm is used as the active polymer layer, wherein the porosity in the active polymer layer is 1 to 90% and the pore size is 100 to 4000 μm; (2) Prepare an absorbent layer on the active polymer layer. The absorbent layer is selected from at least one of a hydrophilic fiber layer, an alginate layer, a semi-permeable polyurethane membrane, and a hydrocolloid layer to obtain an active polymer layer. -absorbent layer; (3) Spray human serum albumin, silk fibroin, thrombin and fibrin solutions evenly on the surface of the active polymer layer-absorbent layer in sequence to form a mesenchymal stem cell attachment layer to obtain a degradable polymer tissue engineering scaffold. 2. The preparation method according to claim 1, wherein the chitin derivative is selected from any one of hydroxy acids, linear saturated aliphatic acid anhydrides, branched chain aliphatic unsaturated acid anhydrides and cyclic acid anhydrides. ; The mixture of chitin derivatives is a combination of two or more chitin derivatives, or a chitin derivative and other biocompatible, biodegradable polymers, and the polymer is selected from polylactide , at least one of polyethylene glycol and 3-hydroxybutyric acid polyester. 3. The preparation method according to claim 2, wherein the hydroxy acid is lactic acid or glycolic acid; the acid group chain length of the linear saturated aliphatic anhydride is C2 to C8, and is selected from acetic anhydride to Any one of octenoic anhydride, acetic acid-propionic anhydride and butyric acid-propionic anhydride; the branched aliphatic unsaturated acid anhydride is methacrylic anhydride or 2-butenoic anhydride; the cyclic acid anhydride is selected from Any one of maleic anhydride, glutaric anhydride, phthalic anhydride and phthalic anhydride. 4. The preparation method according to claim 1, wherein in step (3), the spraying process includes the following steps: adding human serum albumin with a concentration of 1ug/mL to 100mg/mL, 0.1 -100mg/mL silk fibroin mixed solution, spray evenly on the surface of the polymer layer-absorbent layer at a flow rate of 0.1~2mL/s for 5~60s. After the material of this layer is basically dry, spray it uniformly at a flow rate of 0.1~2mL/s for 5~60s. Solution containing 0.1~000U/mL human thrombin), after an interval of 5min~5h, spray a solution containing 0.2~5.0mg/mL fibrinogen at a constant flow rate of 0.1~2mL/s for 5~60s, and let stand at 37℃ for 5min~ 5h, dry. 5. The degradable polymer tissue engineering scaffold obtained by the preparation method according to any one of claims 1 to 4. 6. Application of the degradable polymer tissue engineering scaffold according to claim 5 in the preparation of drugs or drug carriers for regeneration and remodeling of damaged wounds. 7. A composite tissue engineering scaffold system, characterized in that the system includes the degradable polymer tissue engineering scaffold of claim 5 and stem cells loaded and combined on the degradable polymer tissue engineering scaffold. 8. Application of the composite tissue engineering scaffold system according to claim 7 in the preparation of wound healing drugs and/or skin and tissue regeneration drugs.