CN106474565A - A kind of tussah silk fibroin is combined the preparation method of maltodextrin active scaffold material - Google Patents

Abstract

The invention provides a method for preparing an active support material of tussah silk fibroin protein compounded with maltodextrin. The composite support material uses tussah silk fibroin protein and maltodextrin as main raw materials. Tussah silk fibroin can promote the adhesion of fibroblasts; maltodextrin has the functions of material modifier, porogen, cell nutrient, and neutrophil chemoattractant; the preparation process of the material uses phase separation-ultrasound The leaching pore-forming technology is used to adjust the pore size distribution and porosity of the material, which is beneficial to flexibly adjust the mechanical properties and degradation properties of the material, and realize the matching of the degradation rate of the scaffold with the cell growth rate; the preparation process of the scaffold material of the present invention is simple, and the production cycle is short. It has high clinical application value, and is especially suitable for the preparation of artificial skin.

Description

A preparation method of tussah silk fibroin protein compound maltodextrin active scaffold material

technical field

The invention relates to the field of tissue engineering scaffold materials, in particular to a method for preparing an active scaffold material compounded with tussah silk fibroin and maltodextrin.

Background technique

Tissue engineering is to make degradable biomaterials into scaffolds of specific shape and pore size, under the action of growth factors, combined with living cells from specific tissue sources, pre-build a biologically active implant after in vitro culture, and then implant it into the body , to repair or replace defective tissues or organs, and finally form corresponding tissues or organs and perform corresponding engineering. Therefore, the core of tissue engineering is living cells and scaffolding materials for cells to carry out life activities, as well as their mutual use. Scaffold material is the most basic structure of tissue engineered tissue. It provides cells with a place to obtain nutrients, gas exchange, excrete waste and grow metabolism. It is also the material basis for the formation of new tissues and organs with shape and function. Dimensional structures such as cell cultures are fundamentally different. The industrialization of scaffold materials is a prerequisite for the industrialization of tissue engineering.

Tissue engineering has higher and higher requirements for biological materials: (1) Good biological safety, non-toxic, non-allergenic and non-carcinogenic to the human body. (2) Good biocompatibility, no adverse reaction to human body, tissue, blood and immune system. (3) It should have good cell adhesion ability and be able to simulate the physiological environment suitable for cell growth, proliferation and differentiation as much as possible. (4) It must be biodegradable. The degradation products should be non-toxic and able to be metabolized or eliminated by the body. The degradation time should match the healing process and regeneration process. It should have mechanical properties suitable for operation. The change of mechanical properties after degradation should be consistent with the healing process. or regeneration process match. (5) It should have suitable permeability and expected processability, can be shaped arbitrarily according to the formation of tissues and organs, have a specific three-dimensional geometric shape, have a certain pore size and an interconnected three-dimensional structure. (6) The bracket must have certain flexibility and can be organically and closely combined with the body.

Tissue-engineered skin is one of the medical methods for treating skin wounds and defects. Its materials come from a wide range of sources, and the clinical application effect is good. There are pure natural scaffold materials, synthetic scaffold materials, and composite scaffold materials for the material selection. But in general, pure natural scaffold materials are still more dominant and dominant.

Silkworms are divided into two types: domestic silkworm (mulberry silkworm) and wild silkworm (tussah silkworm, celestial silkworm, castor silkworm, etc.). So far, only silkworm cocoons and tussah silkworm cocoons have been industrially utilized. In most researches or reports on silk fibroin materials Among them, the raw materials used are silkworm silk.

Tussah silk is composed of 18 kinds of amino acids, but recent studies have shown that tussah silk threonin contains arginine-glycine-aspartic acid (RGD) tripeptide sequence, RGD sequence acts as a cell integrin receptor and extracellular The recognition site combined with the ligand, the interaction between the street cells and the extracellular matrix and the cells can promote the adhesion of the scaffold to the cells, so that the adhesion rate and value-added rate of the cells on the tussah silk film are much better than those of the mulberry The adhesion rate and value-added rate on the silk fibroin film are regarded as a new type of biomedical material that can be used for the human body. Because the primary structure of tussah silk fibroin contains more cell-specific adhesion sequences RGD, but the silkworm does not contain RGD sequences, the biological performance of tussah silk fibroin-based tissue engineering scaffold materials may be better than that of silkworm silk fibroin. better.

The patent document with the publication number CN101274109A discloses a method for preparing tussah silk fibroin-high polymer porous intertransfer grid support material by foaming method, but the method uses synthetic high polymer, which leads to the failure of the support in the human body. The degradation cycle is difficult to control and adjust, and it is not widely used in clinical practice. Synthetic polymers are biologically inert, have greater predictable properties, batch-to-batch consistency, and are uniquely advanced for specific uses, making these implants often patient-to-patient and site-to-site The difference is smaller than that of enzymatic polymer implants, but there are defects such as hydrophobicity and poor cell adhesion, and some biological signal chains with biological activity are mixed with synthetic polymers to form hybrid materials. improve these deficiencies. However, the physical, chemical, biological and biomechanical properties of synthetic polymer materials will change over time, and the degradation products produced will also have different levels of tissue compatibility with raw materials. Therefore, the biocompatibility of synthetic polymers also needs time. test.

The patent document with publication number 100551449A discloses a preparation method of tussah silk fibroin biomedical material. The invention utilizes tussah silk fibroin protein solution to prepare a three-dimensional non-woven mesh structure material by electrospinning. However, due to the generally high degree of orientation and crystallinity of fibrous silk fibroin, the degradation cycle in vivo may be longer, which is not conducive to the application of tissue engineering skin scaffolds with a shorter degradation cycle. Moreover, this method uses the electrospinning method, which has high requirements on the spinning solution and equipment process conditions, which is not conducive to flexible adjustment of product performance.

For skin scaffolds, the characteristics of the materials used should be fully considered. Scaffold materials are significantly different from general engineering materials, because of their metabolic function, clinical trials of skin substitutes have proved that the most ideal scaffold material should be a physiologically active protein composite material consistent with the structure and composition of human skin, so as to better Well adapted to the complex physiological requirements of the skin.

Maltodextrin is a low-molecular-weight starch derivative, which is often used in the food industry and pharmaceutical excipients. It has good moisture retention, nutritional value, and the effect of promoting immune cell chemotaxis, and can promote the interaction between cells and materials. Compatibility and wound immune regulation (see patent document CN104027346A), conducive to cell growth and wound healing. Combining maltodextrin and tussah silk fibroin can not only promote cell adhesion and wound healing, but also the component maltodextrin in the preparation process acts as a porogen. The component content, porosity and crystallinity of tussah silk fibroin can be adjusted, so that the performance adjustment of the composite scaffold is simple and flexible.

In the patent document with the announcement number 103223192A, a method for preparing a composite material for bone tissue engineering by blending silk fibroin and cyclodextrin and then freeze-drying is introduced, but the material has not been proved Cyclodextrins have immunochemotactic activity.

There are many methods for preparing porous materials for tissue engineering, including freeze-drying, porogen (salting out, leaching), foaming, electrospinning, and phase separation. However, these porogenic methods are all one-time porogenic and have poor flexibility; moreover, the finished products prepared by the porogen method reported so far do not contain porogen, and are often not environmentally degradable, which increases the burden on environmental protection. For example: the patent document with the publication number CN105457096A discloses a preparation method of a biocompatible and degradable tussah fibroin protein tissue engineering scaffold material. The invention introduces the freeze-drying method, porogen method and electrospinning There are three preparation methods, but they are all one-time pore-forming, poor flexibility, and most of the degradable polymers blended with silk fibroin are synthetic polymers, which are not only non-nutritional and immune-promoting, but also have an adjustable degradation cycle. Sex is difficult to meet clinical needs. Therefore, providing a degradable and easy-to-regulate composite scaffold material for artificial skin has become a technical problem to be solved urgently in this field.

Contents of the invention

In view of the above problems, the present invention provides an active porous composite material for tissue engineering skin scaffolds whose degradation performance of tussah silk fibroin protein can be easily adjusted and its preparation method combining phase separation molding, pore formation and secondary pore formation by ultrasonic leaching , the present invention is realized like this:

A preparation method of tussah silk fibroin protein compound maltodextrin active scaffold material, characterized in that, the specific steps are as follows:

(a) Prepare compound preparation solution:

Dissolve the degummed tussah silkworm cocoon shell in the composite aqueous solution of calcium chloride and formic acid, after dialysis with deionized water, concentrate the tussah silk fibroin protein aqueous solution with a mass concentration of 8-10%, and then add maltodextrin into the solution , after stirring and dissolving at room temperature, the composite preparation solution is obtained;

Wherein, the mass ratio of tussah silk fibroin and maltodextrin in the composite preparation liquid is 5:1-4;

(b) Phase separation forming porosity:

Immersing the composite preparation liquid in the phase change forming agent for 4-8 hours to solidify the composite preparation liquid and then place it in a vacuum drying oven at 40°C for vacuum drying to obtain a porous scaffold material;

The phase change forming agent is one of ethanol, acetone, propanol;

(c) Secondary pore formation by ultrasonic leaching:

Submerge the porous scaffold material in an ethanol aqueous solution with a volume concentration of 20-50% and ultrasonically soak for 10-60s to dissolve part of the maltodextrin, realize the second pore forming of the porous composite scaffold, and reduce the crystallinity of the material, and then The material is placed in a vacuum drying oven at 40° C. for vacuum drying to obtain the tussah silk fibroin protein-complexed maltodextrin active scaffold material.

(d) Preservation: put the tussah silk fibroin protein compound maltodextrin active scaffold material into a vacuum sealing bag and seal it, store it in a refrigerator at 0-8°C, and reduce the crystallinity of the material,

Furthermore, in the preparation method of tussah silk fibroin protein composite maltodextrin active scaffold material of the present invention, the average molecular weight of tussah silk fibroin protein in the tussah silk fibroin protein aqueous solution in step (a) is 20-200KDa.

Furthermore, in the preparation method of tussah silk fibroin protein composite maltodextrin active scaffold material of the present invention, the DE value of maltodextrin in step (a) is 10-20.

Furthermore, in the preparation method of tussah fibroin protein composite maltodextrin active scaffold material of the present invention, the mass ratio of tussah silk fibroin protein to maltodextrin in the composite preparation solution in step (a) is 11-171:9.

Further, in the preparation method of tussah silk protein composite maltodextrin active scaffold material of the present invention, the maltodextrin in step (a) is medicinal maltodextrin.

Further, in the preparation method of tussah silk fibroin protein compound maltodextrin active scaffold material of the present invention, the ultrasonic power in step (c) is 25-40KHz. .

Furthermore, in the preparation method of the tussah fibroin protein-based active composite scaffold material of the present invention, the porosity of the tussah silk fibroin protein-based active composite scaffold material obtained in step (c) is 75-95%.

Furthermore, in the preparation method of the tussah fibroin protein-based active composite scaffold material of the present invention, the pore diameter of the tussah silk fibroin protein-based active composite scaffold material obtained in step (c) is 10-500 mm.

Compared with the prior art, the present invention has the following beneficial effects:

1. In the present invention, low molecular weight maltodextrin is compounded in the tussah silk fibroin-based scaffolding material. Maltodextrin is a polysaccharide starch derivative and a neutrophil chemoattractant. By controlling its dosage and pore-forming dissolution rate, it can not only adjust the composition and performance of the composite scaffold, but also act as a porogen, and also have certain cell nutrition and immune regulation effects.

2. After the composite preparation solution in step (a) of the present invention is phase-separated into a porous material in an organic coagulation bath, the secondary pore-forming technology of ultrasonic leaching is used. This method is beneficial to improve the porosity and permeability of the scaffold material; at the same time, this method can adjust the crystallinity of tussah silk fibroin in the semi-finished composite scaffold through the length of ultrasonic leaching time, which is beneficial to the degradation cycle of the scaffold material and wound healing. The healing cycle is matched, and the performance of the scaffold can be flexibly adjusted, which shortens the production cycle of the traditional preparation method from solution preparation to finished product manufacturing.

3. The components of the composite scaffold material of the present invention are all derived from natural macromolecules, can be degraded and absorbed in vivo, and have good biocompatibility. And the porogen maltodextrin as one of the components is a starch derivative, which is easy to be degraded by the environment and has no environmental burden.

4. The preparation process of the present invention is relatively simple, environmentally friendly, low in toxicity, low in energy consumption, and short in production cycle, which is conducive to industrialized mass production and emergency production.

detailed description

The technical idea of the present invention will be further described through the following examples. The reagents and materials involved in the examples are purchased from commercial channels unless otherwise specified.

The medicinal maltodextrin used in the examples was purchased from Shanxi Xiwang Pharmaceutical Co., Ltd., with a DE value of 10-20.

Example 1

1. Prepare compound preparation solution:

Boil the dried tussah cocoon shells in 0.02M sodium carbonate to remove sericin, then dissolve the degummed cocoon shells in a ternary mixed solution of calcium chloride, formic acid, and water with a molar ratio of 1:3:15, After dialysis with deionized water in a dialysis membrane with a molecular weight of 12000, filter to remove insoluble impurities, and concentrate to obtain an aqueous solution of tussah silk fibroin protein with a mass ratio of 8%; then, dissolve medicinal maltodextrin powder in the above solution, and stir Obtain the composite preparation solution evenly.

The mass ratio of tussah silk fibroin protein to medicinal maltodextrin in the compound preparation liquid is 11:9.

2. Phase separation forming and porosity:

Pour 10mL of the composite preparation solution into a Φ35mm×12mm (diameter×height) mold, then place it in ethanol for molding, cause pores for 6 hours, and then dry it in a vacuum oven at 40°C to obtain a porous scaffold material.

3. Ultrasonic secondary leaching causes pores:

Immerse the porous scaffold material in an aqueous ethanol solution with a volume concentration of 50%, and ultrasonicate for 60s at a frequency of 25kHz to dissolve part of the maltodextrin, increase the porosity, and reduce the crystallinity; then dry it in a vacuum oven at 40°C. The tussah silk fibroin-based active composite scaffold material is obtained.

4. Seal the obtained tussah fibroin protein-based active composite scaffold material in a vacuum bag and store it in a refrigerator at low temperature.

Example 2

1. Prepare compound compound preparation solution:

Boil the dried cocoon shells of tussah silkworms with 0.02M sodium carbonate to remove sericin, and then dissolve the degummed cocoon shells in a ternary mixed solution of calcium chloride, formic acid, and water with a molar ratio of 1:2:12. After dialysis with deionized water in a 12000 dialysis membrane, filter to remove insoluble impurities, and concentrate to obtain an aqueous solution of tussah silk fibroin protein with a mass fraction of 8%. Then, the maltodextrin powder is dissolved in the above solution to prepare a composite preparation solution.

The mass ratio of tussah silk fibroin protein to medicinal maltodextrin in the compound preparation liquid is 100:9.

2. Phase separation forming and porosity:

Pour 10mL of composite preparation solution into a Φ35mm×12mm mold, then place it in acetone for molding, cause holes for 4h, and vacuum dry at 40°C.

3. Ultrasonic secondary leaching causes pores:

The porous scaffold material obtained after vacuum drying at 40°C was immersed in an ethanol aqueous solution with a volume concentration of 30%, ultrasonicated for 30s at a frequency of 28kHz to dissolve part of the maltodextrin, and then the material was placed in a vacuum drying oven at 40°C After drying, the tussah silk fibroin-based active composite scaffold material is obtained.

Example 3

1. Prepare compound compound preparation solution:

Boil the dried tussah silkworm cocoons with 0.02M sodium carbonate to remove sericin, and then dissolve the degummed cocoon shells in a ternary mixed solution of calcium chloride, formic acid, and water with a molar ratio of 1:3:12. After dialysis with deionized water in a 3500 dialysis membrane, filter to remove insoluble impurities, and concentrate to obtain an aqueous solution of tussah silk fibroin protein with a mass ratio of 10%; then, dissolve maltodextrin powder in the above solution to prepare a compound Preparation solution.

The mass ratio of tussah silk fibroin to medicinal maltodextrin in the compound preparation liquid is 171:9.

2. Phase separation forming and porosity:

Pour 5mL of the composite preparation solution into a Φ35mm×12mm mold, then place it in propanol for molding, cause pores for 8 hours, and then vacuum-dry at 40°C to obtain a porous scaffold material.

3. Ultrasonic secondary leaching causes pores:

Immerse the obtained porous scaffold material in an aqueous ethanol solution with a volume concentration of 20%, ultrasonicate for 10s at a frequency of 40kHz to dissolve part of the maltodextrin, and then place it at 40°C for vacuum drying to obtain the tussah silk fibroin-based activity Composite scaffold material.

In the specific operation process, the mass ratio of tussah silk fibroin protein to maltodextrin in the compound preparation liquid can also be selected within the range of 11-171:9, both of which can achieve the purpose of the present invention.

Claims (9)

1. a kind of tussah silk fibroin is combined the preparation method of maltodextrin active scaffold material it is characterised in that concrete steps As follows：

（a）Prepare compound preparation solution：

By tussah silk peptide dried cocoon shell after the sodium carbonate liquor of 0.02M boils 30min degumming, it is dissolved in the compound of calcium chloride and formic acid In aqueous solution, after deionized water dialysis, it is concentrated to give the tussah silk fibroin aqueous solution that mass concentration is 8-10%, then Maltodextrin is added in above-mentioned solution, obtains the compound preparation solution of tussah silk fibroin and maltodextrin；

（b）Separated molding pore：

Compound preparation solution is immersed in phase transformation forming agent, submergence 4-8h, it is subsequently placed in 40 DEG C of vacuum drying, obtain cavernous Frame material；

Described phase transformation forming agent is one of ethanol, acetone, propanol；

（c）The secondary pore of ultrasonic leaching：

Cavernous timbering material is immersed in ultrasonic immersion 10-60s in the ethanol water that volumetric concentration is 20-50%, then It is placed in 40 DEG C of vacuum drying, that is, obtain described tussah silk fibroin and be combined maltodextrin active scaffold material.

2. tussah silk fibroin is combined the preparation method of maltodextrin active scaffold material, its feature according to claim 1 It is, described step（a）Tussah silk fibroin mean molecule quantity in middle tussah silk fibroin aqueous solution is 20-200KDa.

3. tussah silk fibroin is combined the preparation method of maltodextrin active scaffold material, its feature according to claim 2 It is, step（a）The DE value of middle maltodextrin is 10-20.

4. it is combined the preparation method of maltodextrin active scaffold material according to the described tussah silk fibroin of one of claim 1-3, It is characterized in that, in the compound aqueous solution of described calcium chloride and formic acid, the mol ratio of calcium chloride, formic acid and water is followed successively by 1：2-3: 12-15.

5. it is combined the preparation method of maltodextrin active scaffold material according to the described tussah silk fibroin of one of claim 1-3, It is characterized in that, step（a）In described compound preparation solution, tussah silk fibroin is 11-171 with the mass ratio of maltodextrin:9.

6. it is combined the preparation method of maltodextrin active scaffold material according to the described tussah silk fibroin of one of claim 1-3, It is characterized in that, step（a）Described maltodextrin is medicinal maltodextrin.

7. tussah silk fibroin is combined the preparation method of maltodextrin active scaffold material, its feature according to claim 6 It is, step（c）Ultrasonic power is 25-40KHz.

8. it is combined the preparation method of maltodextrin active scaffold material according to the described tussah silk fibroin of one of claim 1-3, It is characterized in that, step（c）The porosity of the tussah silk fibroin base activity compound support frame material obtaining is 75-95%.

9. tussah silk fibroin is combined the preparation method of maltodextrin active scaffold material, its feature according to claim 8 It is, step（c）The aperture of the tussah silk fibroin base activity compound support frame material obtaining is 10-500mm.