CN110302432A - A kind of preparation method of the full thickness skin tissue engineering bracket with graded pore structure - Google Patents

A kind of preparation method of the full thickness skin tissue engineering bracket with graded pore structure Download PDF

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CN110302432A
CN110302432A CN201910513466.4A CN201910513466A CN110302432A CN 110302432 A CN110302432 A CN 110302432A CN 201910513466 A CN201910513466 A CN 201910513466A CN 110302432 A CN110302432 A CN 110302432A
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gel
bracket
printing
full thickness
tissue engineering
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CN110302432B (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

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  • Dermatology (AREA)
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Abstract

The present invention relates to organization engineering skin technical fields, the preparation method of specially a kind of full thickness skin tissue engineering bracket with graded pore structure, hybrid inorganic-organic materials are prepared for using sol-gel technique exploration, according to the structure and composition characteristics of skin histology, select CS-Si-Gel component as skin corium, CS-Si component successfully constructs the full thickness skin tissue engineering bracket with graded pore structure using more nozzle printing systems as epidermis.Selected materials of the present invention have excellent biocompatibility, antibiotic property etc., and prepared bracket has the advantage of bionic structure.In addition, carry out layering printing using 3-D scanning modeling technique, successively stack, it can be achieved that personalized customization, to improve the success rate of full thickness dermal reparation.Its research that also can be used for vitro skin model will play a significant role in terms of drug and cosmetics test and tumor research.

Description

A kind of preparation method of the full thickness skin tissue engineering bracket with graded pore structure
Technical field
The present invention relates to the technical fields of organization engineering skin, refer in particular to a kind of full thickness skin with graded pore structure The preparation method of tissue engineering bracket.
Background technique
Skin can be divided into epidermis, skin corium and subcutaneous tissue as the maximum organ of human body in anatomical structure, And contain accessory organ (sweat gland, leather bag) and blood vessel, lymphatic vessel, muscle and nerve etc..Since its large area is exposed to outside In environment, various damages are highly prone to, ulcer as caused by burn, operative incision and chronic disease (diabetes etc.) etc. adds Skin texture complexity, the healing of the surface of a wound is usually extremely difficult.
Although the skin wound dressing applied at present can accelerate wound healing, for large area, deep dermis defect, Its reparation and regeneration that skin histology is difficult to realize in structure and function.
With the development of organizational project, organization engineering skin bracket has become the important channel of defect of skin reparation.For Full thickness skin damage, since the substitute that skin corium is difficult to regenerate, therefore can only repair epidermis is difficult to meet condition.Holostrome skin Skin tissue engineering rack provides new thinking to solve this problem.Full thickness skin tissue engineering bracket, was both replaced comprising epidermis For object, also contain skin corium substitute, wherein the anti-microbial property of epidermis can reduce the inflammatory reaction in agglutination, and true Cortex substitute can stimulate the regeneration of dermis layer tissue.The bracket avoids secondary epidermic grafting and performs the operation and alleviate patient's pain It is bitter.
Chitosan is the positively charged polycation polysaccharide of nature one kind few in number, knot as biomaterial Structure and property are similar to the main component glycosaminoglycan of extracellular matrix, have excellent biocompatibility, film forming, antibacterial Property.But as timbering material, there are mechanical strength deficiency, degradation rate and tissue generating rates to mismatch for single chitosan The disadvantages of.
Element silicon is element important in human body, and there are some researches prove the Si ion discharged from material can promote blood vessel Change and fibroblastic proliferation.When the hybrid material with the random network of chitosan/silica is as skin callus When material is implanted into, under humoral effect, Si-O-Si key is destroyed, and soluble silicon release, it is new that local Si concentrations raising can promote cell Old metabolism excites the autocrine mediated response of the wound healing factor, promotes the proliferation of angiogenesis and fibrocyte, to greatly promote Into the reparation process of skin.
Forming agent of the organic-inorganic material using organic component as hybridization network is combined into organic and inorganic component not There is the entirety of phase interface, the uniformity of resulting materials is good, and transparency is high, while having excellent mechanical performance.It is solidifying by colloidal sol- Glue method, which prepares chitosan-silicon dioxide hybrid material, can improve material property, to meet the good flexibility of Graftskin, elasticity Requirement, and the reparation of skin can be greatly facilitated.
Conventional stent forming method such as particle leaching method, phase separation method, freeze-drying, gas foaming method, fibrage Method etc., it is difficult to realize pore structure be precisely controlled and personalized customization, so that it is complicated to be difficult to meet skin tissue engineering multilayer The requirement of structure.
3D printing can be realized as a kind of technology for constructing 3D solid based on the layer-by-layer mode for stacking build-up materials The integration building of bracket complex topography and internal microstructure.Since the epidermis of skin is mainly fine and close cuticula, corium Layer contains more blood vessel and sweat gland, lower compared to epidermis density.There is gradient aperture skin by 3D printing technique preparation Skin tissue engineering rack, high porosity and bionic gradient aperture can realize effective transport of nutriment and metabolic waste, And can promote tissue vascularization process, to further realize the reparation of defect of skin.
Summary of the invention
The purpose of the present invention is to overcome the shortcomings of the existing technology and deficiency, proposes a kind of with the complete of graded pore structure The preparation method of layer skin tissue engineering scaffold, this method selected materials have excellent biocompatibility, antibiotic property etc., made Standby bracket has the advantage of bionic structure.In addition, carrying out layering printing using 3-D scanning modeling technique, successively stack, it can be real Existing personalized customization, to improve the success rate of full thickness dermal reparation.Its research that also can be used for vitro skin model, It will play a significant role in terms of drug and cosmetics test and tumor research.
To achieve the above object, a kind of technical solution provided by the present invention are as follows: full thickness skin with graded pore structure The preparation method of tissue engineering bracket, the bracket share two layers, respectively epidermis and skin corium, wherein the epidermis is adopted With chitosan/silica dioxide gel production, i.e. CS-Si gel makes, and the skin corium is solidifying using chitosan/silica/gelatin Glue production, i.e. CS-Si-Gel gel make；The preparation process of the bracket is as follows:
It prepares CS-Si gel: chitosan is dissolved in the chitosan for preparing 8-12wt% in the aqueous acetic acid of 4%-10% Solution；Then the TEOS solution hydrolyzed in advance is added, stirring is transferred to it after mixing in printing barrel, and de-bubble is placed on Gel 2-48h at room temperature；
Preparation CS-Si-Gel gel: it is 10-15wt% gelatin solution that gelatin, which is add to deionized water compound concentration, GPTMS is added into gelatin solution, stirs 1-2h under 50 DEG C of -60 DEG C of water bath conditions, prepares modified gelatin solution A；Weigh shell Glycan is scattered in the chitosan suspension B that 8-12wt% is prepared in deionized water；It measures TEOS solution and is dissolved in the acidity that pH value is 2-3 In deionized water solution, TEOS hydrolyzate C is made；Modified gelatin solution A is uniformly mixed with chitosan suspension B, is then added Enter TEOS hydrolyzate C, being sufficiently stirred is uniformly mixed three, so that gelatin is sufficiently handed over chitosan/silicon dioxide hybrid materials Obtained colloidal sol is transferred in printing barrel, removes bubble, finally gel 12-24h at room temperature by connection；
3D printing full thickness skin tissue engineering bracket: it is prepared using more nozzle printing systems, printing step are as follows: will CS-Si gel and CS-Si-Gel gel are loaded into different printing heads, install different needle sizes, skin corium needle diameter 200-400 μm, 100-200 μm of epidermis needle diameter, calibrator (-ter) unit；Default threedimensional model is imported, moulded dimension height is set, Epidermis printing height is 0.5-1.5mm, and skin corium printing height is 1-3mm；Pad parameter, epidermis fiber spacing are set 50-100 μm, 100 μm -400 μm, print speed 20-30mm/s of skin corium fiber spacing, print air pressure 0.45-0.5MPa, syringe needle Raise distance 0.4-0.6mm；It is 15-25 DEG C, 0~5 DEG C of platform temperature that nozzle temperature, which is arranged,；Wherein, exist for different materials In print procedure, preforming bracket is obtained after printing to obtain the fiber of uniformly continuous by adjusting air pressure and print speed；
The post-processing of preforming bracket: the bracket of printing shaping being first placed at -20 DEG C and -80 DEG C and distinguishes pre-freeze for 24 hours, It places it in again and is freeze-dried 24-48h in freeze dryer；It is then dipped in alkaline solution and removes acetic acid extra in material, And it is washed repeatedly with a large amount of deionized waters to neutrality；It finally places a stent into -20 DEG C of refrigerators after pre-freeze, is transferred to freeze-drying It is lyophilized completely in machine, immerses crosslinking agent and carry out further crosslinking Treatment, can finally obtain finished product.
Further, the mass ratio of the CS-Si gel is CS/Si=6/1-10/1.
Further, the mass ratio of the CS-Si-Gel gel is CS/Si/Gel=6/1/1-10/1/1.
Further, TEOS and acetic acid molar ratio are 1/8-1/4 in the TEOS solution hydrolyzed in advance, are stirred at room temperature To its complete hydrolysis.
Further, mass ratio of the GPTMS additional amount relative to Gel are as follows: GPTMS/Gel=1/10-3/10.
Further, the immersion alkaline solution is 0.1-0.5M Tris or saturation Na2HPO4Solution or 10% ammonium hydroxide.
Further, the crosslinking agent is EDC and NHS or Geniposide or glutamine transaminage, be crosslinked to bracket anti- It answers.
Further, the skin corium aperture is 100-400 μm, and the epidermis aperture is 50-100 μm.
Compared with prior art, the present invention have the following advantages that with the utility model has the advantages that
1, preparation process of the present invention is precisely easily-controllable, and stable product quality is, it can be achieved that personalized customization.
2, more nozzle printing system preparations applied by invention use more spray head printing alternates, compared to single nozzle before Printing technique is more flexible, and the gradient integrally chemical conversion of different materials can be realized by adjusting corresponding layer height and fiber spacing Type.
3, obtained bracket has skin biomimetic features, and skin corium aperture is 100-400 μm, and epidermis aperture is 50- 100μm.The interface cohesion of the bracket of different gradient-structures is close, has complete integral structure.The freeze-drying skill of use Art is conducive to the transport of nutriment and metabolite so that scaffold fibers have the 10-100 μm of micropore largely penetrated through.Gradient Pore structure can accommodate epidermal cell and corium fibroblast simultaneously, have more superiority than single layer skin tissue engineering scaffold.
4, the chitosan used has been used for skin, nerve, in osteochondral tissue engineering as timbering material and carrier, The antiphlogistic antibacterial effect having and anti-tumor activity are able to suppress microorganism growth, accelerating wound healing.The present invention passes through colloidal sol- Gel method, not only with chitosan ionic bonding occurs for TEOS hydrolysis gained silane, but also is formed by hydrogen bond action and chitosan Interpenetrating networks.The Si ion that hybrid material is dissolved out can promote vascularization and fibroblastic proliferation, to greatly promote Into the reparation process of skin.Meanwhile hybrid material shows splendid elasticity and water absorption and swelling performance, is conducive to keep disease Aqueous nutritional ingredient necessary to stove peripheral cell is grown.The present invention further creates in chitosan/SiO 2 hybrid network It is new to introduce gelatin, as dermal layer of the skin substitute.Hydrolysate of the gelatin as collagen, still remains in structure The active group of collagen has many excellent performances, such as good biocompatibility, degradability, plasticity, stronger hydrophilic Property and reactivity.The adherency for being greatly facilitated corium fibroblast is proliferated by the components of gelatin of introducing, the regeneration for skin corium It is of great significance.
In conclusion the present invention beats chitosan/silica/gelatin hybrid material and 3D relative to research before Print combines, and is prepared for the full thickness skin tissue engineering bracket with graded pore structure, has for the reparation of skin full-thickness defects Significance.
Detailed description of the invention
Fig. 1 is chitosan in Examples 1 and 2/silica/gelatin (CS-Si-Gel) gel difference gelatine content bracket Graph of stretch performance.
Fig. 2 is rheological behaviour storage modulus (G') of the CS-Si in 25 DEG C of sol-gel transitions in embodiment 1 and 3 With Loss modulus (G ") with the variation diagram of angular speed.
Fig. 3 is the full thickness skin tissue engineering bracket optical photograph with graded pore structure prepared by embodiment 2.
Specific embodiment
Below with reference to multiple specific embodiments, the invention will be further described.
Embodiment 1
With the full thickness skin tissue engineering bracket of graded pore structure prepared by the present embodiment, two layers is shared, respectively Epidermis and skin corium, wherein the epidermis is made of chitosan/silica dioxide gel production, i.e. CS-Si gel, this is true Cortex is made of chitosan/silica/gelatin gel production, i.e. CS-Si-Gel gel；The preparation process of the bracket is such as Under:
The preparation of chitosan-silicon dioxide hydridization (CS-Si) gel: 8g chitosan is dissolved in 4% aqueous acetic acid and is made The chitosan solution of standby 8wt%；What addition hydrolyzed in advance contains 0.8g SiO2TEOS solution, stirring make its be uniformly mixed, will Gained colloidal sol is transferred in barrel, de-bubble postposition gel 48h at room temperature.
Gelatine-chitosan-SiO 2 hybrid (10Gel-CS-Si) gel preparation: by 1.2g gelatin (Gel) addition go from Compound concentration is 10wt% gelatin solution A in sub- water, and 0.12g GPTMS, play under 50 DEG C of water bath conditions are added into above-mentioned solution Strong stirring 2h；It weighs 12g chitosan (CS) and is scattered in the chitosan suspension B for preparing 8wt% in deionized water；Measure TEOS solution It is dissolved in the acid deionized water solution that pH value is 2, TEOS hydrolyzate C is made.
Modified gelatin solution A is uniformly mixed with chitosan suspension B, is then added and contains 1.2g SiO2TEOS water Liquid C is solved, being sufficiently stirred is uniformly mixed three, so that gelatin and chitosan/silicon dioxide hybrid materials are full cross-linked.By gained To colloidal sol be transferred in printing barrel, remove bubble, finally gel is for 24 hours at room temperature.
3D printing full thickness skin tissue engineering bracket: it is prepared using more nozzle printing systems, printing step are as follows: by two Kind material is loaded into different printing heads, installs different needle sizes, 100 μm of epidermis needle diameter, skin corium syringe needle is straight 200 μm of diameter, calibrator (-ter) unit；Default threedimensional model is imported, moulded dimension is set, wherein epidermis printing height is 0.5mm, corium Layer printing height is 1mm；Pad parameter is set, wherein 50 μm of epidermis fiber spacing, 100 μm of skin corium fiber spacing, printing Speed 20-30mm/s, prints air pressure 0.45-0.5MPa, and syringe needle raises distance 0.4-0.6mm；It is 15 DEG C that nozzle temperature, which is arranged, is put down 0 DEG C of platform temperature；Wherein, for different materials in print procedure, by adjusting air pressure and print speed, uniformly connected with obtaining Continuous fiber；Preforming bracket is obtained after printing.
The post-processing of preforming bracket: the bracket of printing shaping being first placed at -20 DEG C and -80 DEG C and distinguishes pre-freeze for 24 hours, It places it in again and is freeze-dried 48h in freeze dryer；Later, it is dipped in 10% ammonium hydroxide and removes acetic acid extra in material, be used in combination A large amount of deionized waters are washed repeatedly to neutrality；It finally places a stent into -20 DEG C of refrigerators after pre-freeze, is transferred in freeze drier Freeze-drying completely immerses crosslinking agent EDC and NHS and carries out further crosslinking Treatment, can obtain finished product.
Embodiment 2
The preparation process of the full thickness skin tissue engineering bracket with graded pore structure of the present embodiment is as follows:
The preparation of chitosan-silicon dioxide hydridization (CS-Si) gel: 12g chitosan is dissolved in 10% aqueous acetic acid Prepare the chitosan solution of 12wt%；What addition hydrolyzed in advance contains 2g SiO2TEOS solution, stirring make its be uniformly mixed, Gained colloidal sol is transferred in barrel, de-bubble postposition gel 48h at room temperature.
The preparation of gelatine-chitosan-SiO 2 hybrid (20Gel-CS-Si) gel: 2g gelatin (Gel) is added to 8g Compound concentration is 20wt% gelatin solution A in ionized water, and 0.2g GPTMS, play under 50 DEG C of water bath conditions are added into above-mentioned solution Strong stirring 2h；It weighs 12g chitosan (CS) and is scattered in the chitosan suspension B for preparing 8wt% in deionized water；Measure TEOS solution It is dissolved in the acid deionized water solution that pH value is 3, TEOS hydrolyzate C is made.
Modified gelatin solution A is uniformly mixed with chitosan suspension B, is then added and contains 1.2g SiO2TEOS water Liquid C is solved, being sufficiently stirred is uniformly mixed three, so that gelatin and chitosan/silicon dioxide hybrid materials are full cross-linked.By gained To colloidal sol be transferred in printing barrel, remove bubble, finally gel is for 24 hours at room temperature.
3D printing full thickness skin tissue engineering bracket: it is prepared using more nozzle printing systems, printing step are as follows: by two Kind material is loaded into different printing heads, installs different needle sizes, 200 μm of epidermis needle diameter, skin corium syringe needle is straight 400 μm of diameter, calibrator (-ter) unit；Default threedimensional model is imported, moulded dimension is set, wherein epidermis printing height is 1.5mm, corium Layer printing height is 3mm；Pad parameter is set, wherein 100 μm of epidermis fiber spacing, 400 μm of skin corium fiber spacing, printing Speed 20-30mm/s, prints air pressure 0.45-0.5MPa, and syringe needle raises distance 0.4-0.6mm；It is 25 DEG C that nozzle temperature, which is arranged, is put down 5 DEG C of platform temperature；Wherein, for different materials in print procedure, by adjusting air pressure and print speed, uniformly connected with obtaining Continuous fiber；Preforming bracket is obtained after printing.
The post-processing of preforming bracket: the bracket of printing shaping being first placed at -20 DEG C and -80 DEG C and distinguishes pre-freeze for 24 hours, It places it in again and is freeze-dried 48h in freeze dryer；Later, it is dipped in saturation Na2HPO4Solution removes vinegar extra in material Acid, and washed repeatedly with a large amount of deionized waters to neutrality；It finally places a stent into -20 DEG C of refrigerators after pre-freeze, immerses crosslinking agent Geniposide carries out further crosslinking Treatment, and finished product can be obtained by being transferred in freeze drier freeze-drying completely.
Embodiment 3
The preparation process of the full thickness skin tissue engineering bracket with graded pore structure of the present embodiment is as follows:
The preparation of chitosan-silicon dioxide hydridization (CS-Si) gel: 8g chitosan is dissolved in 4% aqueous acetic acid and is made The chitosan solution of standby 8wt%；The TEOS solution hydrolyzed in advance is added, stirring is uniformly mixed it, gained colloidal sol is transferred to In barrel, de-bubble postposition gel 2h at room temperature.
The preparation of gelatine-chitosan-SiO 2 hybrid (10Gel-CS-Si) gel: deionization is added in 2g gelatin (Gel) Compound concentration is 10wt% gelatin solution A in water, and 0.6g GPTMS is added into above-mentioned solution, acutely stirs under 50 DEG C of water bath conditions Mix 2h；It weighs 12g chitosan (CS) and is scattered in the chitosan suspension B for preparing 8wt% in deionized water；TEOS solution is measured to be dissolved in In the acid deionized water solution that pH value is 2, TEOS hydrolyzate C is made.
Modified gelatin solution A is uniformly mixed with chitosan suspension B, is then added and contains 2g SiO2TEOS hydrolysis Liquid C, being sufficiently stirred is uniformly mixed three, so that gelatin and chitosan/silicon dioxide hybrid materials are full cross-linked.It will be acquired Colloidal sol be transferred in printing barrel, remove bubble, finally gel 12h at room temperature
3D printing full thickness skin tissue engineering bracket: it is prepared using more nozzle printing systems, printing step are as follows: by two Kind material is loaded into different printing heads, installs different needle sizes, 100 μm of epidermis needle diameter, skin corium syringe needle is straight 200 μm of diameter, calibrator (-ter) unit；Default threedimensional model is imported, moulded dimension is set, wherein epidermis printing height is 0.5mm, corium Layer printing height is 1mm；Pad parameter is set, wherein 50 μm of epidermis fiber spacing, 100 μm of skin corium fiber spacing, printing Speed 20-30mm/s, prints air pressure 0.45-0.5MPa, and syringe needle raises distance 0.4-0.6mm；It is 15 DEG C that nozzle temperature, which is arranged, is put down 0 DEG C of platform temperature；Wherein, for different materials in print procedure, by adjusting air pressure and print speed, uniformly connected with obtaining Continuous fiber；Preforming bracket is obtained after printing.
The post-processing of preforming bracket: the bracket of printing shaping being first placed at -20 DEG C and -80 DEG C and distinguishes pre-freeze for 24 hours, It places it in again and is freeze-dried 48h in freeze dryer；Later, it is dipped in 0.5M Tris solution and removes acetic acid extra in material, And it is washed repeatedly with a large amount of deionized waters to neutrality；It finally places a stent into -20 DEG C of refrigerators after pre-freeze, is transferred to freeze-drying It is lyophilized completely in machine, immerses in glutamine transaminage and carry out further crosslinking Treatment, finished product can be obtained.
(a), (b) are the tensile strength and elongation at break corresponding in fracture with bracket, 20Gel-CS-Si in Fig. 1 0.26 ± 0.02MPa of tensile strength of component bracket, corresponding elongation at break are 38.7%；10Gel-CS-Si and CS-Si group Point, tensile strength is respectively 0.2 ± 0.01MPa and 0.18 ± 0.02MPa, and corresponding elongation at break is respectively 32.3% He 29.1%.When gelatine content increases, bracket tensile strength increases, and elasticity increases, and more meets Graftskin requirement.
The rheological behavior of slurry controllably plays a crucial role the accurate of appearance structure of print carriage.By Fig. 2 It can be seen that, as CS-Si gelation 2h, storage modulus (G') is greater than loss modulus (G "), and it is solidifying to show that slurry has already appeared colloidal sol- Glue transformation；As gelation 48h, with the increase of frequency, G' and G " increase therewith, finally no longer change, and show colloidal sol It has been fully converted to solid gel.Experiment can be according to demand in the selection sol-gel time within the scope of 2-48h.
(a), (b) they are the full thickness skin tissue engineering bracket optical photograph with graded pore structure in Fig. 3, as seen from the figure, Different interlayers with gradient-structure closely connect, and have complete integral structure；Epidermis pore structure is fine and close, skin corium hole Diameter is bigger, achievees the purpose that full thickness skin is bionical.
Embodiment described above is only the preferred embodiments of the invention, and but not intended to limit the scope of the present invention, therefore All shapes according to the present invention change made by principle, should all be included within the scope of protection of the present invention.

Claims (8)

1. a kind of preparation method of the full thickness skin tissue engineering bracket with graded pore structure, it is characterised in that: the bracket Share two layers, respectively epidermis and skin corium, wherein the epidermis is using chitosan/silica dioxide gel production, i.e. CS- The production of Si gel, the skin corium are made of chitosan/silica/gelatin gel production, i.e. CS-Si-Gel gel；It is described The preparation process of bracket is as follows:
It prepares CS-Si gel: chitosan is dissolved in the chitosan solution for preparing 8-12wt% in the aqueous acetic acid of 4%-10%； Then the TEOS solution hydrolyzed in advance is added, stirring is transferred to it after mixing in printing barrel, and de-bubble is placed on room temperature Lower gel 2-48h；
Preparation CS-Si-Gel gel: it is 10-20wt% gelatin solution, Xiang Ming that gelatin, which is add to deionized water compound concentration, GPTMS is added in sol solution, stirs 1-2h under 50 DEG C of -60 DEG C of water bath conditions, prepares modified gelatin solution A；Weigh chitosan It is scattered in the chitosan suspension B that 8-12wt% is prepared in deionized water；Measure TEOS solution be dissolved in pH value be 2-3 acidity go from In sub- aqueous solution, TEOS hydrolyzate C is made；Modified gelatin solution A is uniformly mixed with chitosan suspension B, is then added TEOS hydrolyzate C, being sufficiently stirred is uniformly mixed three, so that gelatin and chitosan/silicon dioxide hybrid materials are full cross-linked, Obtained colloidal sol is transferred in printing barrel, removes bubble, finally gel 12-24h at room temperature；
3D printing full thickness skin tissue engineering bracket: it is prepared using more nozzle printing systems, printing step are as follows: by CS-Si Gel and CS-Si-Gel gel are loaded into different printing heads, install different needle sizes, epidermis needle diameter 100- 200 μm, 200-400 μm of skin corium needle diameter, calibrator (-ter) unit；Default threedimensional model is imported, moulded dimension height, epidermis are set Layer printing height is 0.5-1.5mm, and skin corium printing height is 1-3mm；Pad parameter, epidermis fiber spacing 50-100 are set μm, 100 μm -400 μm, print speed 20-30mm/s of skin corium fiber spacing, print air pressure 0.45-0.5MPa, syringe needle raise away from From 0.4-0.6mm；It is 15-25 DEG C, 0~5 DEG C of platform temperature that nozzle temperature, which is arranged,；Wherein, different materials was being printed Cheng Zhong obtains preforming bracket by adjusting air pressure and print speed to obtain the fiber of uniformly continuous after printing；
The post-processing of preforming bracket: the bracket of printing shaping is first placed at -20 DEG C and -80 DEG C and distinguishes pre-freeze for 24 hours, then will It, which is placed in freeze dryer, is freeze-dried 24-48h；It is then dipped in alkaline solution and removes acetic acid extra in material, be used in combination A large amount of deionized waters are washed repeatedly to neutrality；It finally places a stent into -20 DEG C of refrigerators after pre-freeze, is transferred in freeze drier Freeze-drying completely immerses crosslinking agent and carries out further crosslinking Treatment, can finally obtain finished product.
2. a kind of preparation method of full thickness skin tissue engineering bracket with graded pore structure according to claim 1, It is characterized by: the mass ratio of the CS-Si gel is CS/Si=6/1-10/1.
3. a kind of preparation method of full thickness skin tissue engineering bracket with graded pore structure according to claim 1, It is characterized by: the mass ratio of the CS-Si-Gel gel is CS/Si/Gel=6/1/1-10/1/1.
4. a kind of preparation method of full thickness skin tissue engineering bracket with graded pore structure according to claim 1, It is characterized by: TEOS/ acetic acid molar ratio is 1/8-1/4 in the TEOS solution hydrolyzed in advance, stirred at room temperature to it Complete hydrolysis.
5. a kind of preparation method of full thickness skin tissue engineering bracket with graded pore structure according to claim 1, It is characterized by: mass ratio of the GPTMS additional amount relative to Gel are as follows: GPTMS/Gel=1/10-3/10.
6. a kind of preparation method of full thickness skin tissue engineering bracket with graded pore structure according to claim 1, It is characterized by: immersed alkaline solution is 0.1-0.5M Tris or saturation Na2HPO4Solution or 10% ammonium hydroxide.
7. a kind of preparation method of full thickness skin tissue engineering bracket with graded pore structure according to claim 1, It is characterized by: the crosslinking agent is EDC and NHS or Geniposide or glutamine transaminage, cross-linking reaction is carried out to bracket.
8. a kind of preparation method of full thickness skin tissue engineering bracket with graded pore structure according to claim 1, It is characterized by: the skin corium aperture is 100-400 μm, the epidermis aperture is 50-100 μm.
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