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WO2016090893A1 - Matériau de support d'ingénierie tissulaire à base de réticulation d'éthényle-sulfhydryle et son procédé de préparation - Google Patents

Matériau de support d'ingénierie tissulaire à base de réticulation d'éthényle-sulfhydryle et son procédé de préparation Download PDF

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Publication number
WO2016090893A1
WO2016090893A1 PCT/CN2015/082299 CN2015082299W WO2016090893A1 WO 2016090893 A1 WO2016090893 A1 WO 2016090893A1 CN 2015082299 W CN2015082299 W CN 2015082299W WO 2016090893 A1 WO2016090893 A1 WO 2016090893A1
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WIPO (PCT)
Prior art keywords
vinyl
scaffold material
preparing
cross
tissue engineering
Prior art date
Application number
PCT/CN2015/082299
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English (en)
Chinese (zh)
Inventor
李玲琍
休斯•蒂姆•查尔斯
郝晓娟
陈浩
王磊
Original Assignee
温州医科大学附属眼视光医院
温州生物材料与工程研究所
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Application filed by 温州医科大学附属眼视光医院, 温州生物材料与工程研究所 filed Critical 温州医科大学附属眼视光医院
Publication of WO2016090893A1 publication Critical patent/WO2016090893A1/fr

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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
    • A61L27/14—Macromolecular 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/28—Treatment by wave energy or particle radiation

Definitions

  • the present invention relates to the field of tissue engineering, and in particular to a tissue-based scaffold material based on vinyl-thiol cross-linking and a preparation method thereof.
  • tissue engineering The most basic idea of tissue engineering is to isolate and culture cells in vitro, inoculate a certain amount of cells onto a scaffold with a certain spatial structure, and form a cell with mutual adhesion, growth and secretion, and secretion of extracellular matrix.
  • Material as an artificial extracellular matrix for tissue engineering research (extracellular Matrix, ECM) is an important aspect of tissue engineering research that supports cell berthing, growth, reproduction, metabolism, and formation of new tissues.
  • Tissue engineering scaffold material refers to a material that can bind to tissue living cells and can be implanted into living organisms. It can provide cells with access to nutrients, gas exchange, waste discharge and growth and development, and also form new morphological and functional tissues.
  • the material basis of the organ.
  • the ideal tissue engineering material should have a three-dimensional porous structure, biodegradability, good biocompatibility, plasticity and mechanical strength.
  • For tissue engineering corneas should have transparent refractive, oxygen permeability and other characteristics.
  • Gelatin is a water-soluble protein mixture obtained by hydrolysis of collagen, and its molecular weight is generally between tens of thousands and hundreds of thousands. Gelatin maintains the triple helix structure of collagen and contains arginine-glycine-aspartate (RGD)
  • RGD arginine-glycine-aspartate
  • the sequence which has excellent hydrophilicity and biocompatibility, can promote cell adhesion and growth; at the same time, gelatin removes the immunogenicity of collagen and reduces possible pathogen infection.
  • gelatin has been widely used in the field of tissue engineering. However, gelatin has a large brittleness and degrades rapidly when used alone. Therefore, the strength of gelatin is often increased by chemical crosslinking, and the gelatin degradation time is prolonged.
  • Photocuring cross-linking provides a fast and controllable method of forming a gel network.
  • Light curing cross-linking refers to the use of light
  • the initiator forms a gel by initiating cross-linking curing by visible light or ultraviolet light.
  • the photocuring cross-linking method has the following advantages: the precursor aqueous solution can be cross-linked in situ, and thus can be used for preparing an injectable gel; the product geometry is easy to control; the curing time is short at room temperature or physiological temperature (less than one second) To a few minutes); lower reaction heat etc.
  • the precursor of photopolymerization hydrogel has good fluidity, so it can be used for the preparation of special-shaped repair materials, and has become a research hotspot of current tissue engineering scaffold materials.
  • cartilage tissue engineering techniques can embed chondrocytes in biocompatible, biodegradable scaffolds to complete the transplantation of chondrocytes.
  • Invention patent CN 103157141 A A preparation process of a medical tissue engineering scaffold", by first preparing an elastomer mold of polydimethylsiloxane, and then preparing a single layer of the stent by a solution casting-freeze drying method, and finally obtaining a single sheet by a lamination method.
  • the layer scaffold is fixed to obtain the tissue engineering scaffold, but the tissue engineering scaffold material layer obtained by the method is unstable between layers, and the solvent bonding has a great influence on the biocompatibility of the tissue engineering scaffold material; and the mold is prepared.
  • the predecessor step adds complexity to the process.
  • Patent CN 103520770 A "porous material for tissue engineering scaffold” is made of polycaprolactone and polyethylene oxide as base material, and the intermediate product is obtained by adding micro-cylinder twin-screw extrusion granulation and then microcellular foaming method.
  • the porous scaffold material is obtained by vacuum drying after filtration, but the preparation process is complicated, and the utilization rate of the raw material is reduced by twin-screw extrusion granulation, and the biocompatibility of the obtained porous scaffold material is greatly affected.
  • Patent CN 202654450 U uses bubble electrospinning technology to produce fibrous tissue engineering scaffold materials, but this method requires high equipment, and electrospinning is closely related to the structural properties of the polymer matrix, and can be used for static electricity. The variety of natural polymers that are spun is very limited, and the grasp of the resulting product structure and performance stability is not enough.
  • tissue engineering scaffold material prepared by the invention has good biocompatibility and good mechanical properties, and the preparation method is environmentally friendly, non-polluting, and has a wide range of raw materials.
  • the invention provides a biocompatible tissue engineering scaffold material which can be cross-linked in situ, and can realize on-demand treatment of tissue.
  • the technical solution adopted by the present invention is: a vinyl-sulfonium-based cross-linked tissue engineering scaffold material, which is a vinyl-mercapto-based crosslinked network structure.
  • the invention relates to a vinyl-sulfhydryl-based cross-linked tissue engineering scaffold material, wherein the tissue engineering scaffold material is a biodegradable polymer material with amino and carboxyl groups, such as gelatin, collagen, protein, polypeptide, poly Polysaccharides, etc.
  • a preparation method of a tissue-engineered scaffold material based on vinyl-sulfonium-based cross-linking wherein a vinylation modification reagent and a thiolation modification reagent are respectively used for vinylation modification and thiol modification treatment, and then The raw materials subjected to the vinylation modification treatment and the thiolation modification treatment are thoroughly mixed and photocured and crosslinked under ultraviolet lamp irradiation.
  • the method for preparing a vinyl-sulfonium-based cross-linked tissue engineering scaffold material wherein the vinylation modifying reagent is one of methacrylic anhydride, valeric anhydride, acrylic anhydride, and maleic anhydride.
  • the preparation method of the vinyl-ruthenium-based cross-linked tissue engineering scaffold material is 50 ° C, the reaction environment is alkaline condition, and the pH value is controlled at 7.4. Between 8.
  • the method for preparing a vinyl-sulfonium-based cross-linked tissue engineering scaffold material wherein the vinylation modification treatment has a raw material solution concentration of 5%-20%% w/v.
  • the method for preparing a vinyl-thiol-based cross-linked tissue engineering scaffold material has a rate of addition of a vinylation modification reagent of 0.2 mL/min to 0.5 mL/min, and an addition amount of 0.5% to 1.5. %v/v.
  • the method for preparing a vinyl-sulfonium-based crosslinked tissue engineering scaffold material has a concentration of a raw material solution of the thiolation modification treatment of 0.5% to 2.0% w/v.
  • the pH value of the thiolation modification reaction system described in the preparation method of the vinyl-sulfonium-based crosslinked tissue engineering scaffold material is controlled to be 4.71 - 4.81.
  • the method for preparing a vinyl-sulfonium-based cross-linked tissue engineering scaffold material wherein the reaction temperature of the thiolation modification treatment is 37 °C.
  • the method for preparing a vinyl-sulfonium-based cross-linked tissue engineering scaffold material wherein the vinylation-modified raw material and the thiolated modified raw material are mixed at a mass ratio of 2:1.
  • the method for preparing a vinyl-ruthenium-based cross-linked tissue engineering scaffold material wherein the thiolated modified raw material is subjected to nitrogen-discharging treatment before mixing.
  • the photocuring cross-linking photoinitiator according to the method for preparing a vinyl-sulfonium-based cross-linked tissue engineering scaffold material is a photoinitiator of type 2959, and the addition amount is 0.1%-1.0%. w/v.
  • the method for preparing a vinyl-sulfhydryl-based cross-linked tissue engineering scaffold material has an ultraviolet radiation intensity of 50-300 mW and an irradiation time of 20 s-3 min.
  • the invention has the beneficial effects that the present invention provides a vinyl-ruthenium-based cross-linked tissue engineering scaffold material and a preparation method thereof.
  • the tissue engineering scaffold material prepared by the invention has good biocompatibility, has suitable mechanical strength, can be matched with potential applications, is biodegradable, and can realize on-demand treatment.
  • the invention provides an in-situ ultraviolet curing cross-linking preparation technology, and the preparation process is green, non-polluting and non-toxic.
  • Figure 1 is a schematic diagram of gelatin vinylation modification reaction
  • Figure 2 is a schematic diagram of gelatinization modification reaction of gelatin
  • Figure 3 is a schematic diagram of UV curing crosslinking reaction
  • Figure 4 is a scanning electron micrograph of the surface and section of the tissue engineering scaffold of the present invention
  • Figure 5 is a 3D growth fluorescence microscopic confocal picture of cells in tissue engineering scaffold materials
  • the modified biodegradable polymer material having an amino group and a carboxyl group may be gelatin, collagen, protein, polypeptide, polyglycan, or the like.
  • the vinylation modification reagent used is Methacrylic anhydride (Methacrylic anhydride, MA): First, 20 g of gelatin is dissolved in 200 ml of LDPBS solution, the temperature is adjusted to 50 ° C, and magnetic stirring is performed to obtain a uniformly clear gelatin solution; the pH of the solution is adjusted to make the solution alkaline. Secondly, 2 mL of MA was slowly added to the above solution, and slowly added dropwise at a rate of 0.2 mL/min to 0.5 mL/min. At the same time, control the pH of the whole reaction system, and adjust with 5 M NaOH to stabilize the pH of the entire reaction system. Between 7.4-8, judge with precision PH test paper.
  • MA Methacrylic anhydride
  • the dialysis bag used had a molecular weight cut-off of 1 KDa, dialysis for 5-7 days, and the dialysis solvent was water to remove excess unreacted MA, and the dialysis temperature was set to 50 °C.
  • the dialysis temperature was set to 50 °C.
  • freeze-dry. Fifth, save in the dark.
  • the vinylation modifying agent used may also be one of valer anhydride, acrylic anhydride, and maleic anhydride.
  • EDTA 0.2 mmol/L was added to the dialysate during dialysis to inhibit oxidation of the terminal sulfhydryl group. Dialysis for 5-7 days. Rotary steaming, lyophilization, and preservation in the dark.
  • l2959 - hydroxyethoxy-2-methylpropiophenone
  • 1mL of 75% ethanol solution the dissolved vinylated gelatin is mixed with thiolated gelatin, and 7.5 uL of 2959 ethanol solution is added to the mixture, Vortex was shaken for 3 min; 150 uL of the mixed solution was cast in a mold, placed under ultraviolet light, irradiated for 20 s - 3 min; taken out to obtain a cross-linked tissue engineering scaffold material; stored in PBS solution and stored at 4 ° C.
  • the mold cavity specifications are: ⁇ 15 mm ⁇ 100 um, ⁇ 15 mm ⁇ 200 um.
  • the ultraviolet light intensity is 50-300 mmW.
  • the scaffold obtained by UV-curing cross-linking was immersed in a PBS solution containing a double-antibody for 24 hours, and plated in a 24-well plate with complete medium (90%).
  • F12: DMEM 1:1, 10% FBS, 1% anti-anti, 5 ⁇ g/mL insulin, 10 ng/mLEGF) pre-culture for 24 hours to remove impurities, mouse fibroblasts (1929) were planted on the scaffold material, static culture in vitro for 7 days, electron microscopic observation and MTS test analysis, as shown in Figure 4, see the cell can Good growth on the scaffold and cell survival rate of more than 70%.
  • 100 mg of vinylated gelatin obtained in the first step was dissolved in 1 mL of PBS solution; 50 mg of thiolated gelatin obtained in the second step was dissolved in 500 uL of PBS solution; and the two solutions were respectively placed in boiling water of 100 ° C for 5 min. Under sterile conditions, the two were miscible, and 0.5% of a 1959 ethanol solution was added and mixed well.
  • a 100 uL mixture was taken and mixed with l929 cells at a cell concentration of 20,000 cells/well, vortexed for 2 min; added to a 96-well plate. Irradiated for 1 min under 365 nm ultraviolet light. After 24 hours of culture, it was found by the life-and-death detection device that the survival rate of the cells on the tissue engineering scaffold material was as high as 90% or more.
  • the l929 cells were pre-treated with the fluorescent dye DilC (3); 150 mg of vinylated gelatin obtained in the first step was dissolved in 1 mL of PBS solution; 75 mg of thiolated gelatin obtained in the second step was dissolved in 500 uL of PBS solution; The solution was placed in boiling water at 100 ° C for 5 min. Under sterile conditions, the two were miscible, and 0.5% of a 1959 ethanol solution was added and mixed well. A 100 uL mixture was taken and mixed with the stained l929 cells at a cell concentration of 20,000 cells/well, vortexed for 2 min; and added to a 96-well plate. Irradiated for 1 min under 365 nm ultraviolet light. Then, after 1, 4, and 7 days of culture, the three-dimensional growth of the cells was observed by laser confocal fluorescence microscopy. As shown in Fig. 5, the cells were well grown on the tissue engineering scaffold material.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Dermatology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
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  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
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Abstract

La présente invention concerne un matériau de support d'ingénierie tissulaire à base de réticulation d'éthényle-sulfhydryle et son procédé de préparation ; le matériau de support présente une structure réticulaire à base de réticulation d'éthényle-sulfhydryle ; et le procédé de préparation comprend : la conduite d'un traitement de modification d'éthényle et d'un traitement de modification de sulfhydryle sur une matière première respectivement par l'intermédiaire d'un modificateur d'éthényle et d'un modificateur de sulfhydryle ; et le mélange minutieux des matières premières après le traitement de modification d'éthényle et le traitement de modification de sulfhydryle, et la conduite d'une réticulation par photodurcissement sous irradiation par une lampe ultraviolette.
PCT/CN2015/082299 2014-12-10 2015-06-25 Matériau de support d'ingénierie tissulaire à base de réticulation d'éthényle-sulfhydryle et son procédé de préparation WO2016090893A1 (fr)

Applications Claiming Priority (2)

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CN201410751589.9A CN104548196B (zh) 2014-12-10 2014-12-10 一种基于乙烯基‑巯基交联的组织工程支架材料及其制备方法
CN201410751589.9 2014-12-10

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CN104548196B (zh) * 2014-12-10 2017-10-20 温州医科大学附属眼视光医院 一种基于乙烯基‑巯基交联的组织工程支架材料及其制备方法
CN106049055B (zh) * 2016-06-06 2019-04-09 陕西科技大学 一种高吸湿透湿性海岛型超细纤维合成革基布的制备方法
CN106832386B (zh) * 2017-01-09 2020-04-21 淮阴工学院 一种有机气凝胶及其制备方法和应用
CN110407933A (zh) * 2019-06-26 2019-11-05 江苏悦智生物医药有限公司 接枝产物及其制备方法
CN114874455B (zh) * 2022-02-28 2023-10-27 中国科学院沈阳自动化研究所 一种中性溶解、具有自组装能力和光交联能力的改性胶原和凝胶的构建方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120225101A1 (en) * 2011-03-02 2012-09-06 Kao Weiyuan J Multifunctional in situ polymerized network via thiol-ene and thiol-maleimide chemistry
CN103724455A (zh) * 2013-12-11 2014-04-16 四川大学 一种透明质酸衍生物及其水凝胶的制备方法
CN104548196A (zh) * 2014-12-10 2015-04-29 温州医科大学附属眼视光医院 一种基于乙烯基-巯基交联的组织工程支架材料及其制备方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120225101A1 (en) * 2011-03-02 2012-09-06 Kao Weiyuan J Multifunctional in situ polymerized network via thiol-ene and thiol-maleimide chemistry
CN103724455A (zh) * 2013-12-11 2014-04-16 四川大学 一种透明质酸衍生物及其水凝胶的制备方法
CN104548196A (zh) * 2014-12-10 2015-04-29 温州医科大学附属眼视光医院 一种基于乙烯基-巯基交联的组织工程支架材料及其制备方法

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