CN108553690A - A kind of porous fibroin microballoon and preparation method thereof for mixing strontium - Google Patents

Abstract

The invention discloses a kind of preparation method of porous fibroin microballoon that mixing strontium, the constituent of porous microsphere is fibroin and strontium chloride, and for the porosity of the porous microsphere 80% or more, aperture is 5 40 μm, and strontium chloride weight ratio accounts for 5% the 38% of total weight.Fibroin albumen of the present invention has been commonly used to health food industry as a kind of native protein molecule, will not generate stimulation to human body；Strontium is a kind of indispensable trace element of human body, has the abilities such as osteogenic induction, the effect of Bone Defect Repari can be improved；Main material of the fibroin albumen as cell carrier simultaneously, can simulate the protein ingredient of extracellular matrix；What is be prepared through the invention mixes strontium fibroin porous microsphere, can carry out the sustained release of strontium, effectively overcomes the acquisition growth factor from animal and uses the various problems such as presence.

Description

A strontium-doped porous silk fibroin microsphere and its preparation method

technical field

The invention relates to a method for preparing porous silk fibroin microspheres doped with strontium, which belongs to the technical field of biomedical materials.

Background technique

Bone and cartilage tissue play a very important role in supporting and protecting the human body. The traditional bone repair method is to use autologous bone and allogeneic bone for bone transplantation. This method is not only limited by bone supply resources, but also expensive. The use of allogeneic bone is also prone to adverse reactions such as immune rejection. With the development of tissue engineering and regenerative medicine, it has achieved great success in bone regeneration, and the regenerated bone has normal functions and biological activities. In the process of bone repair, it is necessary to construct three-dimensional scaffolds loaded with cells and growth factors, such as porous sponge scaffolds, fiber mesh scaffolds, and hydrogels. Compared with these scaffold forms, microcarriers have the ability to be directly injected into tissue defects and With the advantage of exempting from surgical procedures, and as a cell carrier, it can expand the three-dimensional culture of cells, increase the cell culture area and maintain cell phenotype. The surface of the porous microcarrier has openings, and the interior has a through-hole structure, which can facilitate cells to enter the interior of the microcarrier and avoid damage to the cells during the culture process. In addition, porous microcarriers can also enhance the transport of cell nutrients and metabolites, provide more specific surface area for cell growth, and greatly increase the number of cell seeding.

Silk fibroin is a natural macromolecular protein composed of 18 kinds of human amino acids. It can be decomposed and re-degraded into monomer amino acids in the body. The degradation products are non-toxic and can be absorbed. Compared with commonly used synthetic materials such as polylactic acid, etc., silk fibroin can mimic the protein components and functions in tissues, and has the advantages of good biocompatibility, degradability, easy processing and modification, and low immunogenicity. meet the requirements of biological materials. Strontium is an important trace element in bone composition, which can promote osteogenesis and inhibit osteoclastosis. Strontium can also promote the differentiation of pluripotent stem cells into osteoblasts, increase bone mineral density, enhance bone hardness, osteoinductive ability and osteoconductivity, ultimately improve bone microstructure and bone quality, and promote new bone regeneration and repair.

Contents of the invention

The present invention aims at the main problems of existing micro-carriers in the application of tissue engineering, such as the unstable properties of the prepared materials and the incompleteness of the research on the structure and morphology, and combines the advantages and characteristics of silk fibroin to provide a kind of microcarrier based on silk fibroin and strontium chloride. The main material is strontium-doped silk fibroin-based porous microspheres prepared by emulsion method combined with freeze-drying technology. The strontium-doped silk fibroin porous microspheres prepared by the invention are mainly used for bone repair, can effectively avoid problems such as cell loss and insufficient bone mass, and enhance support for articular bones. Concrete technical scheme of the present invention is as follows:

The invention discloses a strontium-doped silk fibroin porous microsphere. The composition of the porous microsphere is silk fibroin and strontium chloride. The weight ratio of strontium chloride accounts for 5%-38% of the total weight.

The invention is a method for preparing strontium-doped porous silk fibroin microspheres, and the specific preparation steps adopted are as follows:

1) Shred silkworm cocoons, degumming and dissolving them, then filter, dialyze and concentrate them into silk fibroin solution;

2) Take petroleum ether in a beaker and pre-cool it at -30°C—-50°C for more than 3.5 hours; it can only be made in this temperature range and time, the shape will change if the temperature is too low or too high, and the freezing effect is not good if the time is too short Well, it can't be molded either.

3) Take another petroleum ether in a new beaker, add an emulsifier, and mix the two evenly;

4) Mix 2%-6.5% (w/v) silk fibroin solution and 0.1%-4% (w/v) strontium chloride evenly, add the solution in 3), and stir into a uniform spherical shape; the benefits of strontium doping Because: the current research is mainly to add BMP-2 etc. to the silk fibroin material and stem cells to differentiate into osteoblasts and osteoblasts in vitro, and VEGF and other cytokines related to angiogenesis to improve the therapeutic effect. However, cytokines still face in clinical application: short half-life, difficult to control the release concentration, mainly obtained from animals, high cost, easy to deteriorate, often accompanied by important problems such as immune rejection. Therefore, finding new tissue engineering-derived factors that promote vascularization and osteogenic differentiation of adult stem cells is a research hotspot in repairing bone defects with tissue engineering technology;

5) Pour the solution of 4) into the pre-cooled petroleum ether in 2) quickly, and then place it in a frozen environment to stand for precipitation;

6) Remove the petroleum ether in the solution of 5) in a freezing atmosphere to obtain strontium-doped silk fibroin microspheres containing ice crystals, freeze the silk fibroin microspheres, and then obtain dry strontium-doped porous silk by freeze-drying protein microspheres.

As a further improvement, the mechanical properties of the strontium-doped porous silk fibroin microspheres in step 6) of the present invention are enhanced by subsequent alcohol treatment. Silk fibroin that has not been treated with alcohol will dissolve in water, and this step is beneficial to wash away the residual emulsifier.

As a further improvement, the strontium-doped porous silk fibroin microspheres of the present invention obtain strontium carbonate mineralized porous silk fibroin microspheres with the ability of stable and slow release of strontium ions according to the principle of biomineralization. After mineralization, strontium ions can achieve a sustained release effect, but if put into water without mineralization, strontium ions will be released quickly, and the expected effect cannot be achieved.

Compared with the prior art, the present invention has the following prominent features:

1) Excellent biocompatibility: As a natural protein molecule, silk fibroin has been widely used in the health food industry and will not stimulate the human body;

2) Injectability: Compared with hydrogels and porous sponge scaffolds, silk fibroin microspheres can be directly injected into tissue defects, which can overcome the problems of difficult operation of bone defects caused by special parts and complex structures;

3) Promote bone repair: strontium is an indispensable trace element in the human body, which has the ability to induce osteogenesis and can improve the effect of bone repair; at the same time, silk fibroin, as the main material of cell carriers, can simulate the protein components of extracellular matrix ;

4) The strontium-doped silk fibroin porous microspheres prepared by the present invention can perform sustained release of strontium, effectively overcoming various problems such as obtaining growth factors from animals and using them;

5) Low cost, environmental protection and no pollution.

Detailed ways

The technical solution of the present invention will be further described in detail through specific examples below. The following examples are explanations of the present invention and the present invention is not limited to the following examples.

Example 1

1) Shred silkworm cocoons, degumming and dissolving them, then filter, dialyze and concentrate them into 2% (w/v) silk fibroin solution;

2) Take 450ml of petroleum ether in a 500ml beaker and pre-cool at -50°C for 4h;

3) Take 40ml of petroleum ether in a new 100ml beaker, add 0.7ml of span-80, and mix the two evenly;

4) Mix 10ml of silk fibroin solution and 0.01g of strontium chloride evenly, add the solution in 3), and stir to form a uniform spherical shape;

5) Pour the solution of 4) into the pre-cooled petroleum ether in 2) quickly, and then place it at -50°C to stand for precipitation;

6) Remove the petroleum ether in the solution of 5) in a freezing atmosphere to obtain strontium-doped silk fibroin microspheres containing ice crystals, freeze the silk fibroin microspheres, and then obtain dry strontium-doped porous silk by freeze-drying protein microspheres.

The composition of the obtained porous microsphere is silk fibroin and strontium chloride, the porosity of the porous microsphere is above 80%, the pore diameter is 5 μm-40 μm, and the weight ratio of strontium chloride is 5%-38% of the total weight .

Example 2

1) Shred silkworm cocoons, degumming and dissolving them, then filter, dialyze and concentrate them into 4% (w/v) silk fibroin solution;

2) Take 450ml of petroleum ether in a 500ml beaker and pre-cool at -50°C for 4h;

3) Take 40ml of petroleum ether in a new 100ml beaker, add 0.7ml of span-80, and mix the two evenly;

4) Mix 10ml of silk fibroin solution and 0.01g of strontium chloride evenly, add the solution in 3), and stir to form a uniform spherical shape;

5) Pour the solution of 4) into the pre-cooled petroleum ether in 2) quickly, and then place it at -50°C to stand for precipitation;

6) Remove the petroleum ether in the solution of 5) in a freezing atmosphere to obtain strontium-doped silk fibroin microspheres containing ice crystals, freeze the silk fibroin microspheres, and then obtain dry strontium-doped porous silk by freeze-drying protein microspheres.

Example 3

1) Shred silkworm cocoons, degumming and dissolving them, then filter, dialyze and concentrate them into 6.5% (w/v) silk fibroin solution;

2) Take 450ml of petroleum ether in a 500ml beaker and pre-cool at -50°C for 4h;

3) Take 40ml of petroleum ether in a new 100ml beaker, add 0.7ml of span-80, and mix the two evenly;

4) Mix 10ml of silk fibroin solution and 0.01g of strontium chloride evenly, add the solution in 3), and stir to form a uniform spherical shape;

5) Pour the solution of 4) into the pre-cooled petroleum ether in 2) quickly, and then place it at -50°C to stand for precipitation;

6) Remove the petroleum ether in the solution of 5) in a freezing atmosphere to obtain strontium-doped silk fibroin microspheres containing ice crystals, freeze the silk fibroin microspheres, and then obtain dry strontium-doped porous silk by freeze-drying protein microspheres.

Example 4

1) Shred silkworm cocoons, degumming and dissolving them, then filter, dialyze and concentrate them into 2.5% (w/v) silk fibroin solution;

2) Take 450ml of petroleum ether in a 500ml beaker and pre-cool at -50°C for 4h;

3) Take 40ml of petroleum ether in a new 100ml beaker, add 0.7ml of span-80, and mix the two evenly;

4) Mix 10ml of silk fibroin solution and 0.01g of strontium chloride evenly, add the solution in 3), and stir to form a uniform spherical shape;

5) Pour the solution of 4) into the pre-cooled petroleum ether in 2) quickly, and then place it at -50°C to stand for precipitation;

6) Remove the petroleum ether in the solution of 5) in a freezing atmosphere to obtain strontium-doped silk fibroin microspheres containing ice crystals, freeze the silk fibroin microspheres, and then obtain dry strontium-doped porous silk by freeze-drying protein microspheres.

Example 5

1) Shred silkworm cocoons, degumming and dissolving them, then filter, dialyze and concentrate them into 2.5% (w/v) silk fibroin solution;

2) Take 450ml of petroleum ether in a 500ml beaker and pre-cool at -50°C for 4h;

3) Take 40ml of petroleum ether in a new 100ml beaker, add 0.7ml of span-80, and mix the two evenly;

4) Mix 10ml of silk fibroin solution and 0.05g of strontium chloride evenly, add the solution in 3), and stir to form a uniform spherical shape;

5) Pour the solution of 4) into the pre-cooled petroleum ether in 2) quickly, and then place it at -50°C to stand for precipitation;

6) Remove the petroleum ether in the solution of 5) in a freezing atmosphere to obtain strontium-doped silk fibroin microspheres containing ice crystals, freeze the silk fibroin microspheres, and then obtain dry strontium-doped porous silk by freeze-drying protein microspheres.

Example 6

1) Shred silkworm cocoons, degumming and dissolving them, then filter, dialyze and concentrate them into 2.5% (w/v) silk fibroin solution;

2) Take 450ml of petroleum ether in a 500ml beaker and pre-cool at -50°C for 4h;

3) Take 40ml of petroleum ether in a new 100ml beaker, add 0.7ml of span-80, and mix the two evenly;

4) Mix 10ml of silk fibroin solution and 0.2g of strontium chloride evenly, add the solution in 3), and stir to form a uniform spherical shape;

5) Pour the solution of 4) into the pre-cooled petroleum ether in 2) quickly, and then place it at -50°C to stand for precipitation;

6) Remove the petroleum ether in the solution of 5) in a freezing atmosphere to obtain strontium-doped silk fibroin microspheres containing ice crystals, freeze the silk fibroin microspheres, and then obtain dry strontium-doped porous silk by freeze-drying protein microspheres.

Example 7

1) Shred silkworm cocoons, degumming and dissolving them, then filter, dialyze and concentrate them into 2.5% (w/v) silk fibroin solution;

2) Take 450ml of petroleum ether in a 500ml beaker and pre-cool at -50°C for 4h;

3) Take 40ml of petroleum ether in a new 100ml beaker, add 0.7ml of span-80, and mix the two evenly;

4) Mix 10ml of silk fibroin solution and 0.4g of strontium chloride evenly, add the solution in 3), and stir to form a uniform spherical shape;

5) Pour the solution of 4) into the pre-cooled petroleum ether in 2) quickly, and then place it at -50°C to stand for precipitation;

6) Remove the petroleum ether in the solution of 5) in a freezing atmosphere to obtain strontium-doped silk fibroin microspheres containing ice crystals, freeze the silk fibroin microspheres, and then obtain dry strontium-doped porous silk by freeze-drying protein microspheres.

Example 8

1) Shred silkworm cocoons, degumming and dissolving them, then filter, dialyze and concentrate them into 2.5% (w/v) silk fibroin solution;

2) Take 450ml of petroleum ether in a 500ml beaker and pre-cool at -30°C for 4h;

3) Take 40ml of petroleum ether in a new 100ml beaker, add 0.7ml of span-80, and mix the two evenly;

4) Mix 10ml of silk fibroin solution and 0.2g of strontium chloride evenly, add the solution in 3), and stir to form a uniform spherical shape;

5) Pour the solution of 4) into the pre-cooled petroleum ether in 2) quickly, and then place it at -50°C to stand for precipitation;

6) Remove the petroleum ether in the solution of 5) in a freezing atmosphere to obtain strontium-doped silk fibroin microspheres containing ice crystals, freeze the silk fibroin microspheres, and then obtain dry strontium-doped porous silk by freeze-drying protein microspheres.

The following table provides a comparison between Comparative Example 1-2 and Example 2. Comparative Example 1-2 only changed the technical feature of silk fibroin concentration, and the rest of the technical features are the same as in Example 2. The selection of silk fibroin concentration is important for the technical solution of the present invention.

Concentration of silk fibroin Strontium chloride content Morphological observation Comparative example 1 1% 0.01g Microspheres are prone to collapse and severe adhesion Example 2 4% 0.01g The surface is porous and the interior has holes Comparative example 2 7% 0.01g Pore size and porosity too small

The following table provides a comparison between Comparative Example 3 and Examples 4 and 7. Comparative Example 3 only changed the content of strontium chloride, and the rest of the technical features are the same as in Example 4, so that the selection of the content of strontium chloride can be obtained by observing the morphology. For the importance of the technical solution of the present invention.

Concentration of silk fibroin Strontium chloride content Morphological observation Example 4 2.5% 0.01g The surface is porous and the interior has holes Example 7 2.5% 0.4g The surface is porous and the interior has holes Comparative example 3 2.5% 0.8g Unable to obtain porous microspheres

The following table provides comparison between Comparative Examples 4-5 and Example 5. Comparative Example 3 only changed the technical feature of pre-cooling temperature, and the rest of the technical features are the same as in Example 5, thus obtaining the pre-cooling temperature by morphology observation. The importance of temperature selection for the technical solution of the present invention.

Concentration of silk fibroin Strontium chloride content pre-cooling temperature Morphological observation Comparative example 4 2.5% 0.05g -20°C Unable to obtain porous microspheres Example 5 2.5% 0.05g -50°C The surface is porous and the interior has holes Comparative example 5 2.5% 0.05g -80°C Pore size and porosity too small

Example 9

1) Shred silkworm cocoons, degumming and dissolving them, then filter, dialyze and concentrate them into 2.5% (w/v) silk fibroin solution;

2) Take 450ml of petroleum ether in a 500ml beaker and pre-cool at -50°C for 4h;

3) Take 40ml of petroleum ether in a new 100ml beaker, add 0.7ml of span-80, and mix the two evenly;

4) Mix 10ml of silk fibroin solution and 0.05g of strontium chloride evenly, add the solution in 3), and stir to form a uniform spherical shape;

5) Pour the solution of 4) into the pre-cooled petroleum ether in 2) quickly, and then place it at -50°C to stand for precipitation;

6) Remove the petroleum ether in the solution of 5) in a freezing atmosphere to obtain strontium-doped silk fibroin microspheres containing ice crystals, freeze the silk fibroin microspheres, and then obtain dry strontium-doped porous silk by freeze-drying protein microspheres.

7) Mix the porous silk fibroin microspheres obtained in 6) with NH ₄ HCO ₃ and place them for 2 days, fix them with ethanol for 30 minutes, rinse the ethanol and NH ₄ HCO ₃ with deionized water, and freeze-dry to obtain strontium ion-stabilized Strontium carbonate mineralized silk fibroin porous microspheres with sustained release capability.

Example 10

1) Shred silkworm cocoons, degumming and dissolving them, then filter, dialyze and concentrate them into 2.5% (w/v) silk fibroin solution;

2) Take 450ml of petroleum ether in a 500ml beaker and pre-cool at -50°C for 4h;

3) Take 40ml of petroleum ether in a new 100ml beaker, add 0.7ml of span-80, and mix the two evenly;

4) Mix 10ml of silk fibroin solution and 0.05g of strontium chloride evenly, add the solution in 3), and stir to form a uniform spherical shape;

5) Pour the solution of 4) into the pre-cooled petroleum ether in 2) quickly, and then place it at -50°C to stand for precipitation;

6) Remove the petroleum ether in the solution of 5) in a freezing atmosphere to obtain strontium-doped silk fibroin microspheres containing ice crystals, freeze the silk fibroin microspheres, and then obtain dry strontium-doped porous silk by freeze-drying protein microspheres.

7) The porous silk fibroin microspheres obtained in 6) were mixed and fixed with ethanol for 30 min, the ethanol was rinsed with deionized water, and then freeze-dried to obtain water-insoluble porous silk fibroin microspheres doped with strontium.

Finally, it should be noted that the above examples are only a few specific implementation examples and comparative examples of the present invention. Apparently, the present invention is not limited to the above examples, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

Claims (4)

1. a kind of porous fibroin microballoon for mixing strontium, which is characterized in that the constituent of the porous microsphere is fibroin and chlorination Strontium, the porosity of the porous microsphere is 80% or more, and aperture is 5-40 μm, and strontium chloride weight ratio accounts for the 5%- of total weight 38%.

2. a kind of preparation method of porous fibroin microballoon as described in claim 1 that mixing strontium, which is characterized in that used tool Preparation step is as follows：

1) silk cocoon is shredded, degumming, after dissolving, using filtering, dialyse, to be condensed into 2%-6.5% (w/v) fibroin albumen molten Liquid；

2) take petroleum ether in beaker, at -30 DEG C --- -50 DEG C of precooling 3.5h or more；

3) it takes petroleum ether in new beaker again, emulsifier is added, the two is made to be uniformly mixed；

4) 2%-6.5% (w/v) silk fibroin protein solution, 0.1%-4% (w/v) strontium chloride are uniformly mixed, it is molten in being added 3) Liquid stirs into uniform-spherical；

5) it in the petroleum ether being pre-chilled in pouring into 4) solution 2) rapidly, then is placed in freezing environment and staticly settles；

6) petroleum ether in removing 5) solution under freezing atmosphere obtains and mixes strontium fibroin albumen microballoon containing ice crystal, by the fibroin Protein microsphere is freezed, then can get the dry porous fibroin protein microsphere for mixing strontium by being freeze-dried.

3. the preparation method according to claim 2 for mixing strontium porous fibroin microballoon, which is characterized in that in the step 6) Mix strontium porous fibroin protein microsphere pass through follow-up alcohols processing enhance its mechanical property.

4. the preparation method according to claim 2 or 3 for mixing strontium porous fibroin microballoon, which is characterized in that described mixes strontium Porous fibroin protein microsphere obtains the fibroin of the strontium carbonate mineralising with strontium ion stable sustained-release ability according to biomineralization principle Albumen porous microsphere.