CN110302432B - Preparation method of full-layer skin tissue engineering scaffold with gradient pore structure - Google Patents

Preparation method of full-layer skin tissue engineering scaffold with gradient pore structure Download PDF

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CN110302432B
CN110302432B CN201910513466.4A CN201910513466A CN110302432B CN 110302432 B CN110302432 B CN 110302432B CN 201910513466 A CN201910513466 A CN 201910513466A CN 110302432 B CN110302432 B CN 110302432B
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skin tissue
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CN110302432A (en
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赵娜如
许珊
董怡帆
王迎军
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South China University of Technology SCUT
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/025—Other specific inorganic materials not covered by A61L27/04 - A61L27/12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/222—Gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y10/00—Processes of additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y70/00—Materials specially adapted for additive manufacturing

Abstract

The invention relates to the technical field of tissue engineering skin, in particular to a preparation method of a full-layer skin tissue engineering scaffold with a gradient pore structure. The selected material has excellent biocompatibility, antibacterial property and the like, and the prepared scaffold has the advantage of bionic structure. In addition, the three-dimensional scanning modeling technology is adopted for layered printing, and the layers are stacked layer by layer, so that personalized customization can be realized, and the success rate of full-layer skin defect repair is improved. It can also be used for the research of in vitro skin models, and plays an important role in the aspects of drug and cosmetic tests and tumor research.

Description

Preparation method of full-layer skin tissue engineering scaffold with gradient pore structure
Technical Field
The invention relates to the technical field of tissue engineering skin, in particular to a preparation method of a full-layer skin tissue engineering scaffold with a gradient pore structure.
Background
The skin is the largest organ of the human body, and is anatomically divided into epidermis, dermis, and subcutaneous tissue, and contains accessory organs (sweat glands, skin sacs) and blood vessels, lymphatic vessels, muscles, nerves, and the like. Because of its large area exposed to the external environment, it is very vulnerable to various injuries, such as burns, surgical incisions and ulcers caused by chronic diseases (diabetes, etc.), and the complex structure of skin tissue, the healing of the wound surface is often very difficult.
The skin wound dressing applied at present can accelerate wound healing, but is difficult to realize skin tissue repair and regeneration on the aspects of structure and function for large-area and deep skin defects.
With the development of tissue engineering, tissue engineering skin scaffolds have become an important approach for skin defect repair. For full-thickness skin lesions, it is difficult to satisfy the condition that only the epidermis layer can be repaired, since the dermis layer is difficult to regenerate. The full-layer skin tissue engineering scaffold provides a new idea for solving the problem. The full-layer skin tissue engineering scaffold comprises an epidermal layer substitute and a dermal layer substitute, wherein the antibacterial property of the epidermal layer can reduce inflammatory reaction in a healing process, and the dermal layer substitute can stimulate regeneration of dermal layer tissues. The bracket avoids secondary epidermal grafting operation and relieves the pain of patients.
The chitosan is used as a biological material, is a cationic polysaccharide with a positive charge in a small number of natural world, has the structure and the property similar to the main component glycosaminoglycan of extracellular matrix, and has excellent biocompatibility, film-forming property and antibacterial property. However, as a scaffold material, single chitosan has the defects of insufficient mechanical strength, mismatched degradation rate and tissue generation rate and the like.
Silicon is an important element in human body, and researches prove that Si ions released from the material can promote vascularization and proliferation of fibroblasts. When the hybrid material with the chitosan/silicon dioxide irregular network is implanted as a skin wound healing material, under the action of body fluid, Si-O-Si bonds are broken, soluble silicon is released, the local Si concentration is increased, the cell metabolism can be promoted, the autocrine reaction of wound healing factors is stimulated, the angiogenesis and the proliferation of fiber cells are promoted, and thus, the skin repair process is greatly promoted.
The organic-inorganic material takes the organic component as a forming agent of a hybrid network, so that the organic component and the inorganic component are combined into a whole without a phase interface, and the obtained material has good uniformity, high transparency and excellent mechanical properties. The chitosan-silicon dioxide hybrid material prepared by the sol-gel method can improve the material performance, so as to meet the requirements of good flexibility and elasticity of the skin substitute, and greatly promote the repair of the skin.
The traditional scaffold forming methods such as a particle leaching method, a phase separation method, a freeze drying method, a gas foaming method, a fiber weaving method and the like are difficult to realize the precise control and the personalized customization of the pore structure, so that the requirements of a multi-layer complex structure of skin tissue engineering are difficult to meet.
3D printing is used as a technology for constructing a three-dimensional entity based on a mode of stacking and accumulating materials layer by layer, and can realize the integrated construction of the complex appearance and the internal microstructure of a support. Since the epidermis layer of the skin is mainly a dense stratum corneum, the dermis layer contains more blood vessels and sweat glands and is less dense than the epidermis layer. The skin tissue engineering scaffold with the gradient aperture is prepared by a 3D printing technology, the high porosity and the bionic gradient aperture can realize effective transportation of nutrient substances and metabolic wastes, and can promote the process of tissue vascularization, thereby further realizing the repair of skin defects.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a preparation method of a full-layer skin tissue engineering scaffold with a gradient pore structure. In addition, the three-dimensional scanning modeling technology is adopted for layered printing, and the layers are stacked layer by layer, so that personalized customization can be realized, and the success rate of full-layer skin defect repair is improved. It can also be used for the research of in vitro skin models, and plays an important role in the aspects of drug and cosmetic tests and tumor research.
In order to achieve the purpose, the technical scheme provided by the invention is as follows: a method for preparing a full-layer skin tissue engineering scaffold with a gradient pore structure comprises two layers, namely an epidermal layer and a dermal layer, wherein the epidermal layer is made of chitosan/silicon dioxide Gel, namely CS-Si Gel, and the dermal layer is made of chitosan/silicon dioxide/gelatin Gel, namely CS-Si-Gel; the preparation process of the scaffold comprises the following steps:
preparing CS-Si gel: dissolving chitosan in 4-10% acetic acid water solution to prepare 8-12 wt% chitosan solution; then adding a pre-hydrolyzed TEOS solution, stirring to uniformly mix the solution, transferring the mixture into a printing material cylinder, removing bubbles, and placing the mixture at room temperature for gelation for 2-48 h;
preparing CS-Si-Gel: adding gelatin into deionized water to prepare a gelatin solution with the concentration of 10-15 wt%, adding GPTMS into the gelatin solution, and stirring for 1-2h under the water bath condition of 50-60 ℃ to prepare a modified gelatin solution A; weighing chitosan, dispersing the chitosan in deionized water to prepare 8-12 wt% of chitosan suspension B; measuring TEOS solution, and dissolving in acidic deionized water solution with pH of 2-3 to obtain TEOS hydrolysate C; uniformly mixing the modified gelatin solution A and the chitosan suspension B, then adding the TEOS hydrolysate C, fully stirring to uniformly mix the modified gelatin solution A, the TEOS hydrolysate C, the TEOS;
3D printing a full-layer skin tissue engineering scaffold: the preparation is carried out by adopting a multi-nozzle printing system, and the printing steps are as follows: loading CS-Si Gel and CS-Si-Gel in different printing nozzles, installing needles with different sizes, wherein the diameter of the needle in the dermis layer is 200-; leading in a preset three-dimensional model, and setting the size height of the model, wherein the printing height of the epidermal layer is 0.5-1.5mm, and the printing height of the dermal layer is 1-3 mm; setting filling parameters, wherein the fiber spacing of the epidermis layer is 50-100 mu m, the fiber spacing of the dermis layer is 100-400 mu m, the printing speed is 20-30mm/s, the printing air pressure is 0.45-0.5MPa, and the needle head lifting distance is 0.4-0.6 mm; setting the temperature of a spray head to be 15-25 ℃ and the temperature of a platform to be 0-5 ℃; the method comprises the following steps of (1) obtaining uniform and continuous fibers by adjusting air pressure and printing speed in the printing process of different materials, and obtaining a preformed support after printing;
post-processing the preformed bracket: pre-freezing the printed and molded stent at-20 ℃ and-80 ℃ for 24h respectively, and freeze-drying the pre-frozen stent in a freeze dryer for 24-48 h; then immersing the material in alkaline solution to remove redundant acetic acid in the material, and repeatedly washing the material to be neutral by using a large amount of deionized water; and finally, placing the bracket in a refrigerator at the temperature of 20 ℃ below zero for pre-freezing, transferring the bracket to a freeze dryer for complete freeze drying, and immersing the bracket in a cross-linking agent for further cross-linking treatment to obtain a finished product.
Further, the mass ratio of the CS-Si gel is 6/1-10/1.
Further, the mass ratio of the CS-Si-Gel is 6/1/1-10/1/1.
Further, the TEOS and acetic acid molar ratio in the pre-hydrolyzed TEOS solution is 1/8-1/4, and the solution is stirred at room temperature until the solution is completely hydrolyzed.
Further, the mass ratio of the GPTMS addition amount to Gel is as follows: GPTMS/Gel 1/10-3/10.
Further, the immersion alkaline solution is 0.1-0.5M Tris or saturated Na2HPO4Solution or 10% ammonia.
Further, the crosslinking agent is EDC and NHS, or genipin or glutamine transaminase, and the crosslinking reaction is carried out on the scaffold.
Furthermore, the aperture of the dermis layer is 100-400 μm, and the aperture of the epidermis layer is 50-100 μm.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the preparation process is accurate and easy to control, the product quality is stable, and personalized customization can be realized.
2. The multi-nozzle printing system applied by the invention adopts multi-nozzle alternate printing, is more flexible compared with the prior single-nozzle printing technology, and can realize gradient integrated molding of different materials by adjusting corresponding layer height and fiber spacing.
3. The prepared scaffold has a skin bionic structure, the aperture of the dermis layer is 100-400 mu m, and the aperture of the epidermis layer is 50-100 mu m. The interfaces of the supports with different gradient structures are tightly combined, and the supports have a complete integrated structure. The freeze drying technology is adopted, so that a large number of through micropores with the diameter of 10-100 mu m exist in the scaffold fiber, and the transportation of nutrient substances and metabolites is facilitated. The gradient pore structure can simultaneously contain epidermal cells and dermal fibroblasts, and is superior to a single-layer skin tissue engineering scaffold.
4. The chitosan used as a scaffold material and a carrier is already used in skin, nerve and osteochondral tissue engineering, and has the functions of anti-inflammation, anti-bacterial and anti-tumor activities, so that the growth of microorganisms can be inhibited, and the wound healing can be accelerated. According to the invention, through a sol-gel method, silane obtained by TEOS hydrolysis is not only subjected to ion bonding with chitosan, but also forms an interpenetrating network with chitosan through hydrogen bond action. Si ions dissolved out by the hybrid material can promote vascularization and proliferation of fibroblasts, thereby greatly promoting the repair process of the skin. Meanwhile, the hybrid material shows excellent elasticity and water absorption swelling performance, and is beneficial to maintaining the water-based nutrient components necessary for the growth of cells around the focus. The invention further creatively introduces gelatin in the chitosan/silicon dioxide hybrid network as a substitute of the dermal layer of the skin. Gelatin, as a hydrolysate of collagen, retains the active groups of collagen structurally, and has many excellent properties, such as good biocompatibility, degradability, plasticity, strong hydrophilicity and reactivity. The introduced gelatin component can greatly promote the adhesion and proliferation of dermal fibroblasts, and has important significance for the regeneration of the dermal layer.
Compared with the previous research, the invention combines the chitosan/silicon dioxide/gelatin hybrid material with 3D printing to prepare the full-layer skin tissue engineering scaffold with the gradient pore structure, and has important significance for repairing the full-layer defect of the skin.
Drawings
FIG. 1 is a graph of the tensile properties of scaffolds of different gelatin contents of chitosan/silica/gelatin (CS-Si-Gel) gels in examples 1 and 2.
FIG. 2 is a graph of the rheological behavior of CS-Si during sol-gel transition at 25 ℃ as a function of storage modulus (G ') and dissipation modulus (G') with angular velocity for examples 1 and 3.
Fig. 3 is an optical photograph of the full-thickness skin tissue engineering scaffold with the gradient pore structure prepared in example 2.
Detailed Description
The invention is further illustrated below with reference to a number of specific examples.
Example 1
The full-layer skin tissue engineering scaffold with the gradient pore structure prepared in the embodiment has two layers, namely an epidermis layer and a dermis layer, wherein the epidermis layer is made of chitosan/silicon dioxide Gel, namely CS-Si Gel, and the dermis layer is made of chitosan/silicon dioxide/gelatin Gel, namely CS-Si-Gel; the preparation process of the scaffold comprises the following steps:
preparation of chitosan-silica hybrid (CS-Si) gel: 8g of chitosan is dissolved in 4 percent acetic acid aqueous solution to prepare 8 weight percent of chitosan solution; adding prehydrolyzed SiO 0.8g2Stirring the TEOS solution to mix evenly, transferring the obtained sol into a charging barrel, removing bubbles, and placing the sol at room temperature for gelation for 48 h.
Preparation of gelatin-chitosan-silica hybrid (10Gel-CS-Si) Gel: adding 1.2g of gelatin (Gel) into deionized water to prepare a gelatin solution A with the concentration of 10 wt%, adding 0.12g of GPTMS into the solution, and vigorously stirring for 2 hours at the temperature of 50 ℃ in a water bath; weighing 12g of Chitosan (CS) and dispersing in deionized water to prepare 8 wt% of chitosan suspension B; measuring TEOS solution, and dissolving in acidic deionized water solution with pH of 2 to obtain TEOS hydrolysate C.
The modified gelatin solution A was mixed homogeneously with the chitosan suspension B, followed by addition of a solution containing 1.2g of SiO2And fully stirring the TEOS hydrolysate C to uniformly mix the TEOS hydrolysate C with the TEOS hydrolysate C, so that the gelatin and the chitosan/silicon dioxide hybrid material are fully crosslinked. The resulting sol was transferred to a printing cylinder, the bubbles were removed and finally gelled for 24h at room temperature.
3D printing a full-layer skin tissue engineering scaffold: the preparation is carried out by adopting a multi-nozzle printing system, and the printing steps are as follows: loading the two materials in different printing nozzles, installing needles with different sizes, wherein the diameter of the needle of the epidermis layer is 100 microns, the diameter of the needle of the dermis layer is 200 microns, and calibrating equipment; importing a preset three-dimensional model, and setting the size of the model, wherein the printing height of the epidermis layer is 0.5mm, and the printing height of the dermis layer is 1 mm; setting filling parameters, wherein the fiber spacing of the epidermis layer is 50 microns, the fiber spacing of the dermis layer is 100 microns, the printing speed is 20-30mm/s, the printing air pressure is 0.45-0.5MPa, and the needle head lifting distance is 0.4-0.6 mm; setting the temperature of a spray head to be 15 ℃ and the temperature of a platform to be 0 ℃; wherein, for different materials in the printing process, the uniform and continuous fibers are obtained by adjusting the air pressure and the printing speed; and printing to obtain the preformed support.
Post-processing the preformed bracket: pre-freezing the printed and molded stent at-20 ℃ and-80 ℃ for 24h respectively, and then freeze-drying the pre-frozen stent in a freeze dryer for 48 h; then, the material is immersed into 10% ammonia water to remove redundant acetic acid in the material, and is repeatedly washed to be neutral by a large amount of deionized water; and finally, pre-freezing the bracket in a refrigerator at the temperature of-20 ℃, transferring the bracket to a freeze dryer for complete freeze drying, and immersing the bracket in crosslinking agents EDC and NHS for further crosslinking treatment to obtain a finished product.
Example 2
The preparation process of the full-thickness skin tissue engineering scaffold with the gradient pore structure of the embodiment is as follows:
preparation of chitosan-silica hybrid (CS-Si) gel: dissolving 12g of chitosan in 10% acetic acid aqueous solution to prepare 12 wt% chitosan solution; adding a prehydrolyzed solution containing 2g of SiO2Stirring the TEOS solution to mix evenly, transferring the obtained sol into a charging barrel, removing bubbles, and placing the sol at room temperature for gelation for 48 h.
Preparation of gelatin-chitosan-silica hybrid (20Gel-CS-Si) Gel: adding 2g of gelatin (Gel) into 8g of deionized water to prepare a gelatin solution A with the concentration of 20 wt%, adding 0.2g of GPTMS into the solution, and violently stirring for 2 hours at the temperature of 50 ℃ in a water bath; weighing 12g of Chitosan (CS) and dispersing in deionized water to prepare 8 wt% of chitosan suspension B; measuring TEOS solution, and dissolving in acidic deionized water solution with pH value of 3 to obtain TEOS hydrolysate C.
The modified gelatinThe solution A was mixed homogeneously with the chitosan suspension B and subsequently 1.2g SiO in the solution was added2And fully stirring the TEOS hydrolysate C to uniformly mix the TEOS hydrolysate C with the TEOS hydrolysate C, so that the gelatin and the chitosan/silicon dioxide hybrid material are fully crosslinked. The resulting sol was transferred to a printing cylinder, the bubbles were removed and finally gelled for 24h at room temperature.
3D printing a full-layer skin tissue engineering scaffold: the preparation is carried out by adopting a multi-nozzle printing system, and the printing steps are as follows: loading the two materials in different printing nozzles, installing needles with different sizes, wherein the diameter of the needle of the epidermal layer is 200 microns, the diameter of the needle of the dermal layer is 400 microns, and calibrating equipment; importing a preset three-dimensional model, and setting the size of the model, wherein the printing height of the epidermis layer is 1.5mm, and the printing height of the dermis layer is 3 mm; setting filling parameters, wherein the fiber spacing of the epidermis layer is 100 microns, the fiber spacing of the dermis layer is 400 microns, the printing speed is 20-30mm/s, the printing air pressure is 0.45-0.5MPa, and the needle head lifting distance is 0.4-0.6 mm; setting the temperature of a spray head to be 25 ℃ and the temperature of a platform to be 5 ℃; wherein, for different materials in the printing process, the uniform and continuous fibers are obtained by adjusting the air pressure and the printing speed; and printing to obtain the preformed support.
Post-processing the preformed bracket: pre-freezing the printed and molded stent at-20 ℃ and-80 ℃ for 24h respectively, and then freeze-drying the pre-frozen stent in a freeze dryer for 48 h; thereafter, it was immersed in saturated Na2HPO4Removing redundant acetic acid in the material by using the solution, and repeatedly washing the material to be neutral by using a large amount of deionized water; and finally, placing the bracket in a refrigerator at the temperature of 20 ℃ below zero for pre-freezing, immersing the bracket in a crosslinking agent genipin for further crosslinking treatment, and transferring the bracket to a freeze dryer for complete freeze drying to obtain a finished product.
Example 3
The preparation process of the full-thickness skin tissue engineering scaffold with the gradient pore structure of the embodiment is as follows:
preparation of chitosan-silica hybrid (CS-Si) gel: 8g of chitosan is dissolved in 4 percent acetic acid aqueous solution to prepare 8 weight percent of chitosan solution; adding a pre-hydrolyzed TEOS solution, stirring to uniformly mix, transferring the obtained sol into a charging barrel, removing bubbles, and placing at room temperature for gelation for 2 h.
Preparation of gelatin-chitosan-silica hybrid (10Gel-CS-Si) Gel: adding 2g of gelatin (Gel) into deionized water to prepare a gelatin solution A with the concentration of 10 wt%, adding 0.6g of GPTMS into the solution, and vigorously stirring for 2 hours at the temperature of 50 ℃ in a water bath; weighing 12g of Chitosan (CS) and dispersing in deionized water to prepare 8 wt% of chitosan suspension B; measuring TEOS solution, and dissolving in acidic deionized water solution with pH of 2 to obtain TEOS hydrolysate C.
The modified gelatin solution A was mixed homogeneously with the chitosan suspension B, followed by addition of a solution containing 2g of SiO2And fully stirring the TEOS hydrolysate C to uniformly mix the TEOS hydrolysate C with the TEOS hydrolysate C, so that the gelatin and the chitosan/silicon dioxide hybrid material are fully crosslinked. Transferring the obtained sol into a printing cylinder, removing bubbles, and finally gelling at room temperature for 12h
3D printing a full-layer skin tissue engineering scaffold: the preparation is carried out by adopting a multi-nozzle printing system, and the printing steps are as follows: loading the two materials in different printing nozzles, installing needles with different sizes, wherein the diameter of the needle of the epidermis layer is 100 microns, the diameter of the needle of the dermis layer is 200 microns, and calibrating equipment; importing a preset three-dimensional model, and setting the size of the model, wherein the printing height of the epidermis layer is 0.5mm, and the printing height of the dermis layer is 1 mm; setting filling parameters, wherein the fiber spacing of the epidermis layer is 50 microns, the fiber spacing of the dermis layer is 100 microns, the printing speed is 20-30mm/s, the printing air pressure is 0.45-0.5MPa, and the needle head lifting distance is 0.4-0.6 mm; setting the temperature of a spray head to be 15 ℃ and the temperature of a platform to be 0 ℃; wherein, for different materials in the printing process, the uniform and continuous fibers are obtained by adjusting the air pressure and the printing speed; and printing to obtain the preformed support.
Post-processing the preformed bracket: pre-freezing the printed and molded stent at-20 ℃ and-80 ℃ for 24h respectively, and then freeze-drying the pre-frozen stent in a freeze dryer for 48 h; then, the material is immersed into 0.5M Tris solution to remove redundant acetic acid in the material, and is repeatedly washed to be neutral by a large amount of deionized water; and finally, placing the scaffold in a refrigerator at the temperature of-20 ℃ for pre-freezing, transferring the scaffold to a freeze dryer for complete freeze drying, and immersing the scaffold in glutamine transaminase for further crosslinking treatment to obtain a finished product.
In FIG. 1, (a) and (b) are the tensile strength and elongation at break corresponding to the stent at break, the tensile strength of the 20Gel-CS-Si component stent is 0.26 +/-0.02 MPa, and the corresponding elongation at break is 38.7%; 10Gel-CS-Si and CS-Si components, the tensile strength of which is 0.2 plus or minus 0.01MPa and 0.18 plus or minus 0.02MPa respectively, and the corresponding elongation at break is 32.3 percent and 29.1 percent respectively. When the content of the gelatin is increased, the tensile strength of the bracket is increased, the elasticity is increased, and the requirement of a skin substitute is met.
The rheological property of the slurry plays an important role in the accurate control of the morphological structure of the printing support. As can be seen from FIG. 2, when CS-Si is gelled for 2h, the storage modulus (G ') is greater than the loss modulus (G'), indicating that the slurry has undergone a sol-gel transition; when the gel is gelled for 48h, as the frequency is increased, G 'and G' are increased, and finally, no change occurs, which indicates that the sol is completely converted into solid gel. The sol-gel time can be selected in the range of 2-48h according to the requirement in the experiment.
Fig. 3 (a) and (b) are optical photographs of a full-layer skin tissue engineering scaffold with a gradient pore structure, and it can be seen that different layers with a gradient structure are tightly connected and have a complete integrated structure; the pore structure of the epidermis layer is compact, the pore diameter of the dermis layer is larger, and the aim of simulating the whole layer of skin is fulfilled.
The above-mentioned embodiments are merely preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, so that the changes in the shape and principle of the present invention should be covered within the protection scope of the present invention.

Claims (8)

1. A preparation method of a full-layer skin tissue engineering scaffold with a gradient pore structure is characterized by comprising the following steps: the bracket comprises two layers, namely a surface layer and a dermis layer, wherein the surface layer is made of chitosan/silicon dioxide Gel, namely CS-Si Gel, and the dermis layer is made of chitosan/silicon dioxide/gelatin Gel, namely CS-Si-Gel; the preparation process of the scaffold comprises the following steps:
preparing CS-Si gel: dissolving chitosan in 4-10% acetic acid water solution to prepare 8-12 wt% chitosan solution; then adding a pre-hydrolyzed TEOS solution, stirring to uniformly mix the solution, transferring the mixture into a printing material cylinder, removing bubbles, and placing the mixture at room temperature for gelation for 2-48 h;
preparing CS-Si-Gel: adding gelatin into deionized water to prepare a gelatin solution with the concentration of 10-20 wt%, adding GPTMS into the gelatin solution, and stirring for 1-2h under the water bath condition of 50-60 ℃ to prepare a modified gelatin solution A; weighing chitosan, dispersing the chitosan in deionized water to prepare 8-12 wt% of chitosan suspension B; measuring TEOS solution, and dissolving in acidic deionized water solution with pH of 2-3 to obtain TEOS hydrolysate C; uniformly mixing the modified gelatin solution A and the chitosan suspension B, then adding the TEOS hydrolysate C, fully stirring to uniformly mix the modified gelatin solution A, the TEOS hydrolysate C, the TEOS;
3D printing a full-layer skin tissue engineering scaffold: the preparation is carried out by adopting a multi-nozzle printing system, and the printing steps are as follows: loading CS-Si Gel and CS-Si-Gel in different printing nozzles, installing needles with different sizes, wherein the diameter of the needle of the epidermis layer is 100-; leading in a preset three-dimensional model, and setting the size height of the model, wherein the printing height of the epidermal layer is 0.5-1.5mm, and the printing height of the dermal layer is 1-3 mm; setting filling parameters, wherein the fiber spacing of the epidermis layer is 50-100 mu m, the fiber spacing of the dermis layer is 100-400 mu m, the printing speed is 20-30mm/s, the printing air pressure is 0.45-0.5MPa, and the needle head lifting distance is 0.4-0.6 mm; setting the temperature of a spray head to be 15-25 ℃ and the temperature of a platform to be 0-5 ℃; the method comprises the following steps of (1) obtaining uniform and continuous fibers by adjusting air pressure and printing speed in the printing process of different materials, and obtaining a preformed support after printing;
post-processing the preformed bracket: pre-freezing the printed and molded stent at-20 ℃ and-80 ℃ for 24h respectively, and freeze-drying the pre-frozen stent in a freeze dryer for 24-48 h; then immersing the material in alkaline solution to remove redundant acetic acid in the material, and repeatedly washing the material to be neutral by using a large amount of deionized water; and finally, placing the bracket in a refrigerator at the temperature of 20 ℃ below zero for pre-freezing, transferring the bracket to a freeze dryer for complete freeze drying, and immersing the bracket in a cross-linking agent for further cross-linking treatment to obtain a finished product.
2. The method for preparing a full-thickness skin tissue engineering scaffold with a gradient pore structure according to claim 1, wherein the full-thickness skin tissue engineering scaffold comprises the following steps: the mass ratio of the CS-Si gel is 6/1-10/1.
3. The method for preparing a full-thickness skin tissue engineering scaffold with a gradient pore structure according to claim 1, wherein the full-thickness skin tissue engineering scaffold comprises the following steps: the mass ratio of the CS-Si-Gel is 6/1/1-10/1/1.
4. The method for preparing a full-thickness skin tissue engineering scaffold with a gradient pore structure according to claim 1, wherein the full-thickness skin tissue engineering scaffold comprises the following steps: the TEOS/acetic acid molar ratio in the pre-hydrolyzed TEOS solution is 1/8-1/4, and the solution is stirred at room temperature until the solution is completely hydrolyzed.
5. The method for preparing a full-thickness skin tissue engineering scaffold with a gradient pore structure according to claim 1, wherein the full-thickness skin tissue engineering scaffold comprises the following steps: the mass ratio of the GPTMS addition amount to Gel is as follows: GPTMS/Gel 1/10-3/10.
6. The method for preparing a full-thickness skin tissue engineering scaffold with a gradient pore structure according to claim 1, wherein the full-thickness skin tissue engineering scaffold comprises the following steps: the alkaline solution is 0.1-0.5M Tris or saturated Na2HPO4Solution or 10% ammonia.
7. The method for preparing a full-thickness skin tissue engineering scaffold with a gradient pore structure according to claim 1, wherein the full-thickness skin tissue engineering scaffold comprises the following steps: the cross-linking agent is EDC and NHS, or genipin or glutamine transaminase, and carries out cross-linking reaction on the scaffold.
8. The method for preparing a full-thickness skin tissue engineering scaffold with a gradient pore structure according to claim 1, wherein the full-thickness skin tissue engineering scaffold comprises the following steps: the aperture of the dermis layer is 100-400 μm, and the aperture of the epidermis layer is 50-100 μm.
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