WO2016090893A1

WO2016090893A1 - Tissue engineered support material based on ethenyl-sulphydryl crosslink and preparation method thereof

Info

Publication number: WO2016090893A1
Application number: PCT/CN2015/082299
Authority: WO
Inventors: 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
Priority date: 2014-12-10
Filing date: 2015-06-25
Publication date: 2016-06-16
Also published as: CN104548196B; CN104548196A

Abstract

A tissue engineered support material based on ethenyl-sulphydryl crosslink and preparation method thereof; the support material has a reticular structure based on ethenyl-sulphydryl crosslink; and the preparation method comprises: conducting ethenyl modification processing and sulphydryl modification processing on a raw material respectively via an ethenyl modifier and a sulphydryl modifier; and thoroughly mixing the raw materials after the ethenyl modification processing and the sulphydryl modification processing, and conducting photocuring crosslinking under ultraviolet lamp irradiation.

Description

Vinyl-sulfonium-based cross-linked tissue engineering scaffold material and preparation method thereof

Technical field

[0001] 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.

Background technique

[0002] 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. A structure or function of tissue or organ. 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) 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. As an excellent natural biomaterial, 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. For example, damaged cartilage is difficult to repair by itself due to the lack of blood vessels. Currently, the more common treatment is to transplant homologous chondrocytes. However, this method involves surgically transplanting healthy cartilage and is limited by the shape of the cartilage. In contrast, cartilage tissue engineering techniques can embed chondrocytes in biocompatible, biodegradable scaffolds to complete the transplantation of chondrocytes.

In recent years, many scholars at home and abroad have used various methods to prepare tissue engineering scaffold materials, in order to be applied in clinical practice. 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. Invention 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 "a tissue engineering scaffold" 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.

Summary of the invention

[0003] In accordance with the deficiencies in the prior art, it is an object of the present invention to provide a vinyl-sulfhydryl-based cross-linked tissue engineering scaffold material and a method of making same. The 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, the reaction temperature of the vinylation modification treatment 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 tissue-based scaffold material based on a vinyl-thiol cross-linking, wherein the thiol-based modifying agent is cysteamine.

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.

DRAWINGS

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

detailed description:

The invention is further described below by way of examples, but the invention is not limited thereto.

The modified biodegradable polymer material having an amino group and a carboxyl group may be gelatin, collagen, protein, polypeptide, polyglycan, or the like.

Preparation of vinylated gelatin

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. Third, after 3 hours of reaction, dialysis, 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. Fourth, freeze-dry. Fifth, save in the dark.

The vinylation modifying agent used may also be one of valer anhydride, acrylic anhydride, and maleic anhydride.

Modified biodegradable polymer material with amino and carboxyl groups

Preparation of thiolated gelatin

Take 5g gelatin powder, dissolve in 500mL PBS solution, adjust the pH with 5MNaH2PO4 and 5M NaOH, so that the pH value in the system is stable at 4.71-4.81. EDC1.9871g, NHS 0.3683g was added, and the mixture was uniformly mixed, and nitrogen gas was bubbled for 20-30 min to remove oxygen. Subsequently, 0.6712 g of Cysteamine was quickly added, and nitrogen gas bubbling was continued. The reaction was stirred for 12 h and the reaction temperature was controlled to 37 °C. The reaction product was steamed, dialyzed, and dialyzed with water. A small amount of 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.

UV curing cross-linking

200 mg of the prepared vinylated gelatin was dissolved in 1 mL of PBS solution; 100 mg of the prepared thiolated gelatin was dissolved in 50 mL of PBS solution, and the dissolution process was carried out by bubbling nitrogen for 15 minutes; 50 mg of 2-hydroxy-4'-(2) was weighed. - hydroxyethoxy)-2-methylpropiophenone (l2959) is dissolved in 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: φ15 mm×100 um, φ15 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, 5μg/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.

The detailed description is only for the purpose of further clarifying the invention, and is not intended to limit the scope of the present invention, and those skilled in the art can make some non-essential improvements and adjustments to the present invention according to the contents of the above invention, which are all within the scope of the present invention. The scope of the invention is defined by the claims.

Claims

A vinyl-sulfhydryl-based cross-linked tissue engineering scaffold material, characterized in that the tissue engineering scaffold material is a vinyl-mercapto-based crosslinked network structure.

2. The vinyl-sulfonium-based cross-linked tissue engineering scaffold material according to claim 1, wherein the tissue engineering scaffold material is a biodegradable polymer material with an amino group and a carboxyl group.

3. A method for preparing a tissue engineering scaffold material based on vinyl-sulfonium-based cross-linking, characterized in that vinylation modification and thiolation modification reagent are used for vinylation modification and thiolation modification of raw materials After the treatment, the raw materials subjected to the vinylation modification treatment and the thiolation modification treatment are thoroughly mixed and photocured and crosslinked under ultraviolet light irradiation.

4. The method for preparing a vinyl-sulfhydryl-based cross-linked tissue engineering scaffold material according to claim 3, wherein the vinylation modifying reagent is methacrylic anhydride, pentene anhydride, and acrylic acid anhydride. And one of maleic anhydride.

5. The method for preparing a vinyl-sulfonium-based cross-linked tissue engineering scaffold material according to claim 3, wherein the vinylation modification treatment has a reaction temperature of 50 ° C and the reaction environment is alkaline. Condition, PH value is controlled between 7.4-8.

6. The method for preparing a tissue-based scaffold material based on vinyl-mercapto cross-linking according to claim 3, wherein the concentration of the raw material solution of the vinylation modification treatment is 5%-20% w/ v.

7. The method for preparing a vinyl-thiol-based cross-linked tissue engineering scaffold material according to claim 3, wherein the vinylation modification reagent is added at a rate of 0.2 mL/min to 0.5 mL/ Min, the addition amount is 0.5% - 1.5% v / v.

8. The method for preparing a tissue-based scaffold material based on vinyl-mercapto cross-linking according to claim 3, wherein the concentration of the raw material solution of the thiolation modification is 0.5%-2.0% w/v.

9. The method for preparing a tissue-based scaffold material based on vinyl-sulfonium-based cross-linking according to claim 3, wherein the PH value of the thiolation modification treatment system is stably controlled at 4.71 - 4.81.

10. The method for preparing a tissue-based scaffold material based on vinyl-thiol cross-linking according to claim 3, wherein the thiolation modification treatment temperature is 37 °C.

11. The method for preparing a vinyl-sulfhydryl-based cross-linked tissue engineering scaffold material according to claim 3, wherein the thiol-based modifying agent is cysteamine.

12. The method for preparing a vinyl-sulfonium-based crosslinked tissue engineering scaffold material according to claim 3, wherein the mass ratio of the vinylated modified raw material to the thiolated modified raw material is 2 :1.

13. The method for preparing a tissue-based scaffold material based on vinyl-sulfonium-based cross-linking according to claim 3, wherein the thiolated modified raw material is subjected to nitrogen-discharging treatment before mixing.

14. The method for preparing a tissue-based scaffold material based on vinyl-sulfonium-based cross-linking according to claim 3, wherein the photo-curing cross-linking photoinitiator is a photoinitiator of type 2959, the amount of addition 0.1% - 1.0% w/v.

15. The method for preparing a tissue-based scaffold material based on vinyl-thiol cross-linking according to claim 3, wherein the ultraviolet radiation intensity is 50-300 mW, and the irradiation time is 20 s-3 min.