阅读说明：本技术 丝素蛋白/羟基磷灰石复合支架及其制备方法 (Fibroin albumen/hydroxyapatite compound rest and preparation method thereof ) 是由 卿湘城 邵云菲 邵增务 张克勤 彭一中 于 2019-09-26 设计创作，主要内容包括：本申请涉及生物医学领域,具体而言,涉及一种丝素蛋白/羟基磷灰石复合支架及其制备方法。一种丝素蛋白/羟基磷灰石复合支架的制备方法,包括：采用微波对浸泡在SBF溶液中的丝素蛋白支架进行辅助矿化反应,使丝素蛋白支架的表面和内部沉积羟基磷灰石。在微波的作用下,SBF溶液能够吸收微波能量,使得SBF溶液中的离子运动加速,促进矿化反应加速,同时由于各部位离子能量吸收比较均匀,所以矿化比较均匀,从而在丝素蛋白支架上沉积均匀的羟基磷灰石。(This application involves fields of biomedicine, in particular to a kind of fibroin albumen/hydroxyapatite compound rest and preparation method thereof.A kind of preparation method of fibroin albumen/hydroxyapatite compound rest, comprising: auxiliary mineralising reaction is carried out to the silk fibroin bracket being immersed in SBF solution using microwave, makes surface and the inside deposition hydroxyapatite of silk fibroin bracket.Under the action of microwave, SBF solution can absorb microwave energy, so that the ion motion in SBF solution accelerates, mineralising reaction is promoted to accelerate, simultaneously because each position ion energy absorb it is relatively uniform, so mineralising is relatively uniform, thus in silk fibroin bracket depositing homogeneous hydroxyapatite.)

1. a kind of fibroin albumen/hydroxyapatite compound rest preparation method characterized by comprising

Carrying out microwave irradiation to the SBF solution immersed with silk fibroin bracket makes the fibroin albumen branch to carry out mineralising reaction The surface of frame and internal deposition hydroxyapatite.

2. fibroin albumen according to claim 1/hydroxyapatite compound rest preparation method, which is characterized in that

It is described to immersed with silk fibroin bracket SBF solution carry out microwave irradiation the step of include: in closed environment, it is right Microwave irradiation is carried out immersed with the SBF solution of silk fibroin bracket.

3. fibroin albumen according to claim 1 or 2/hydroxyapatite compound rest preparation method, which is characterized in that

Use the single time of microwave irradiation for 14-16S, the number of microwave irradiation is 8-10 times.

4. fibroin albumen according to claim 1/hydroxyapatite compound rest preparation method, which is characterized in that

The silk fibroin bracket is made by the way that silk fibroin solution to be freeze-dried.

5. fibroin albumen according to claim 4/hydroxyapatite compound rest preparation method, which is characterized in that

The silk fibroin solution is the concentration of the silk fibroin solution by the way that the first silk fibroin solution and the second silk fibroin solution to be mixed to get For 1.8-2.2w/v%；

First silk fibroin solution is to be concentrated to get third silk fibroin solution at 56-64 DEG C, the concentration of the third silk fibroin solution For 5.5-6.5w/v%；The concentration of first silk fibroin solution is 18-23w/v%；

Second silk fibroin solution is diluted with water to first silk fibroin solution after concentration is 0.4-0.6w/v% in 55-65 DEG C hatching to formed nanofiber after be made.

6. fibroin albumen according to claim 5/hydroxyapatite compound rest preparation method, which is characterized in that

The third silk fibroin solution is to remove sericin after boiling raw silk to obtain fibroin, then will be dissolved in after fibroin drying After being dissolved in LiBr solution, it is made after filtering, dialysis, centrifugation.

7. fibroin albumen according to claim 6/hydroxyapatite compound rest preparation method, which is characterized in that

It is described raw silk is boiled after remove sericin the step of, comprising:

By raw silk 0.45-0.55w/v% Na₂CO₃40-50min is boiled in solution, is washed out removing sericin.

8. fibroin albumen according to claim 4/hydroxyapatite compound rest preparation method, which is characterized in that

Described the step of being freeze-dried silk fibroin solution, comprising:

After the silk fibroin solution is mixed with glycerol, it is freeze-dried in -19~-21 DEG C, then again freezes first time for the first time Dry first fibroin bracket obtained is soaked in water, after removing glycerol, is carried out second and is freeze-dried, the fibroin egg is made White rami frame.

9. fibroin albumen according to claim 1/hydroxyapatite compound rest preparation method, which is characterized in that

The concentration of the SBF solution is 1 × SBF solution.

10. a kind of fibroin albumen/hydroxyapatite compound rest, which is characterized in that described in any item using claim 1-9 Fibroin albumen/hydroxyapatite compound rest preparation method is made.

Technical field

This application involves field of biomedicine, in particular to a kind of fibroin albumen/hydroxyapatite compound rest and Preparation method.

Background technique

Fibroin albumen/hydroxyapatite compound rest is to utilize bone holder material made from natural raw silk substance.This silk Fibroin/hydroxyapatite compound rest has better biocompatibility and regulatable degradability.

But there are the mineralising time is long when traditional fibroin albumen/hydroxyapatite compound rest preparation at present, low efficiency The problem of.

Summary of the invention

The embodiment of the present application is designed to provide a kind of fibroin albumen/hydroxyapatite compound rest and its preparation side The problem of method, it is long to be intended to improve existing fibroin albumen/hydroxyapatite compound rest mineralising time, low efficiency.

In a first aspect, the application provides a kind of technical solution:

A kind of preparation method of fibroin albumen/hydroxyapatite compound rest, comprising:

Carrying out microwave irradiation to the SBF solution immersed with silk fibroin bracket makes fibroin albumen branch to carry out mineralising reaction The surface of frame and internal deposition hydroxyapatite.

Under the action of microwave, SBF solution can absorb microwave energy, so that the ion motion in SBF solution accelerates, promote Accelerate into mineralising reaction, simultaneously because the absorption of each position ion energy is relatively uniform, so mineralising is relatively uniform, thus in fibroin The hydroxyapatite of depositing homogeneous on albumen bracket.

In the other embodiments of the application, the above-mentioned SBF solution to immersed with silk fibroin bracket carries out microwave irradiation The step of include: in closed environment, to immersed with silk fibroin bracket SBF solution carry out microwave irradiation.

Closed environment is more advantageous to SBF solution and absorbs microwave energy, uniform mineralising.

In the other embodiments of the application, the above-mentioned single time for using microwave irradiation for 14-16S, microwave irradiation Number is 8-10 times.

Within the time of above-mentioned microwave irradiation and the numbers range of microwave irradiation, it can guarantee hydroxyapatite pattern And ingredient.More than above-mentioned range, time too long solution temperature heating is excessively high, influence mineralising hydroxyapatite pattern and at Point, time too short reaction time is inadequate, and deposition effect is poor.

In the other embodiments of the application, above-mentioned silk fibroin bracket is made by the way that silk fibroin solution to be freeze-dried.

In the other embodiments of the application, above-mentioned silk fibroin solution is by by the first silk fibroin solution and the second silk fibroin solution It is mixed to get, the concentration of silk fibroin solution is 1.8-2.2w/v%；

First silk fibroin solution is to be concentrated to get third silk fibroin solution at 56-64 DEG C, and the concentration of third silk fibroin solution is 5.5-6.5w/v%；The concentration of first silk fibroin solution is 18-23w/v%；

Second silk fibroin solution is to be diluted with water to the first silk fibroin solution after concentration is 0.4-0.6w/v% to incubate at 55-65 DEG C Change to after forming nanofiber and is made.

The fibroin bracket with nanofibrous structures is made in hatching, and fibroin bracket is made to have better porous structure, improves The uniformity of hydroxyapatite deposition.

In the other embodiments of the application, above-mentioned third silk fibroin solution is to remove sericin after boiling raw silk to obtain silk Then element will be dissolved in after dissolving in LiBr solution after fibroin drying, be made after filtering, dialysis, centrifugation.

It will be dissolved in LiBr solution after fibroin drying, can fully dissolve fibroin.

In the other embodiments of the application, it is above-mentioned raw silk is boiled after remove sericin the step of, comprising:

By raw silk 0.45-0.55w/v% Na₂CO₃40-50min is boiled in solution, is washed out removing sericin.

Sericin can be effectively removed using above-mentioned solvent.

In the other embodiments of the application, above-mentioned the step of being freeze-dried silk fibroin solution, comprising:

After silk fibroin solution is mixed with glycerol, it is freeze-dried in -19~-21 DEG C, then again freezes first time for the first time Dry first fibroin bracket obtained is soaked in water, after removing glycerol, is carried out second and is freeze-dried, fibroin albumen branch is made Frame.

In the other embodiments of the application, the concentration of above-mentioned SBF solution is 1 × SBF solution.

The dosage of SBF solution is few, saves reagent.

Second aspect, the application provide a kind of technical solution:

A kind of fibroin albumen/hydroxyapatite compound rest, using such as above-mentioned fibroin albumen/hydroxyapatite composite support The preparation method of frame is made.

The fibroin albumen/hydroxyapatite compound rest surface and internal all uniform depositions have hydroxyapatite, uniformly Property is good.

Detailed description of the invention

Technical solution in ord to more clearly illustrate embodiments of the present application, below will be to needed in the embodiment attached Figure is briefly described, it should be understood that the following drawings illustrates only some embodiments of the application, therefore is not construed as pair The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this A little attached drawings obtain other relevant attached drawings.

Fig. 1 is fibroin albumen provided by the embodiments of the present application/hydroxyapatite compound rest preparation flow schematic diagram；

Fig. 2 is fibroin albumen provided by the embodiments of the present application/hydroxyapatite compound rest and tradition SBF mineralising fibroin The scanning electron microscope (SEM) photograph of bracket；

Fig. 3 is fibroin albumen provided by the embodiments of the present application/hydroxyapatite compound rest and tradition SBF mineralising fibroin Bracket mechanical property；

Fig. 4 is fibroin albumen provided by the embodiments of the present application/hydroxyapatite compound rest and tradition SBF mineralising fibroin Bracket heat analysis test chart；

Fig. 5 is fibroin albumen provided by the embodiments of the present application/hydroxyapatite compound rest and tradition SBF mineralising fibroin Scaffold degradation performance；

Fig. 6 is fibroin albumen provided by the embodiments of the present application/hydroxyapatite compound rest and tradition SBF mineralising fibroin The influence that bracket is proliferated BMSC；

Fig. 7 is fibroin albumen provided by the embodiments of the present application/hydroxyapatite compound rest and tradition SBF mineralising fibroin Influence of the bracket to BMSC Osteoblast Differentiation.

Specific embodiment

It is described in detail below in conjunction with embodiment of the embodiment to the application, but those skilled in the art will Understand, the following example is merely to illustrate the application, and is not construed as limitation scope of the present application.It is not specified in embodiment specific Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is The conventional products that can be obtained by commercially available purchase.

In addition, term " first ", " second ", " third " etc. are only used for distinguishing description, it is not understood to indicate or imply Relative importance.

SBF solution (simulated body fluid) is the component of simulated body fluid and a kind of solution of pH value.SBF solution mainly by The composition such as sodium chloride, potassium chloride, dipotassium hydrogen phosphate, magnesium chloride, sodium bicarbonate.Silk fibroin bracket is soaked in SBF solution, phosphorus Mineralising reaction can occur for the phosphate such as sour hydrogen dipotassium, generate hydroxyapatite deposition in silk fibroin bracket.Invention human hair Existing, under the conditions of this, the speed that mineralising reaction occurs for phosphate is slow, and mineralising is uneven, if to improve mineralization velocity, Need the higher SBF solution of concentration.

The application embodiment provides a kind of preparation method of fibroin albumen/hydroxyapatite compound rest, comprising:

Fibroin albumen branch is made to carry out mineralising reaction using microwave irradiation to the SBF solution immersed with silk fibroin bracket The surface of frame and internal deposition hydroxyapatite.

Microwave refers to that frequency is the electromagnetic wave of 300MHz~3000GHz (3THz), and wavelength is between 0.1 millimeter~1 meter Electromagnetic wave, microwave have the characteristic absorbed.Under the action of microwave, SBF solution can absorb microwave energy, so that SBF solution In ion motion accelerate, promote mineralising reaction accelerate, simultaneously because each position ion energy absorb it is relatively uniform, so mineralising It is relatively uniform, thus in the surface of silk fibroin bracket and the hydroxyapatite of internal depositing homogeneous.

Still optionally further, microwave selects frequency for 2000-3000MHz.Still optionally further, microwave select frequency for 2100-2800MHz.For example, selecting frequency for 2200MHz, 2300MHz, 2400MHz, 2500MHz, 2600MHz, 2700MHz Microwave.Referring to Fig.1, in some embodiments of the application, above-mentioned fibroin albumen/hydroxyapatite compound rest side is prepared Method includes:

S1, silk fibroin solution is prepared.

Silk fibroin solution is by the way that the first silk fibroin solution and the second silk fibroin solution to be mixed to get, and the concentration of silk fibroin solution is 1.8-2.2w/v%；

First silk fibroin solution is to be concentrated to get third silk fibroin solution at 56-64 DEG C, and the concentration of third silk fibroin solution is 5.5-6.5w/v%；The concentration of first silk fibroin solution is 18-23w/v%；

Second silk fibroin solution is to be diluted with water to the first silk fibroin solution after concentration is 0.4-0.6w/v% to incubate at 55-65 DEG C Change to after forming nanofiber and is made.

By the way that the second above-mentioned silk fibroin solution in 55-65 DEG C of hatching to formation nanofiber, is enabled to finally be made Silk fibroin bracket have more porous structures mentioned so that the deposition for hydroxyapatite provides more attachment sites High mineralization effect.

By the way that the concentration of the first above-mentioned silk fibroin solution is concentrated into 18-23w/v%, it can guarantee that subsequent first fibroin is molten The concentration of the silk fibroin solution obtained after liquid and the mixing of the second silk fibroin solution is 1.8-2.2w/v%, and then is guaranteed subsequent molten with SBF When liquid mineralising reaction, more attachment sites can be provided for the deposition of hydroxyapatite.

Still optionally further, it is by third silk that third silk fibroin solution is concentrated to get to the first silk fibroin solution at 56-64 DEG C Plain solution is placed in drying box about 24 hours and is concentrated to get the first silk fibroin solution.

Further, third silk fibroin solution is to remove sericin after boiling raw silk to obtain fibroin, then that fibroin is dry After be dissolved in LiBr solution dissolve after, after filtering, dialysis, centrifugation be made.

Raw silk is resulting product after mulberry cocoon filature.Containing about 20% sericin, boiling can remove silk gum after boiling Albumen.

Still optionally further, sericin is removed after raw silk being boiled, comprising:

By raw silk 0.45-0.55w/v% Na₂CO₃40-50min is boiled in solution, is then thoroughly washed with distilled water To remove sericin.

Still optionally further, sericin is removed after raw silk being boiled, comprising:

By raw silk 0.5w/v% Na₂CO₃45min is boiled in solution, is then thoroughly washed with distilled water to remove silk gum Albumen.

Further, the concentration of above-mentioned LiBr solution selects 9.0-9.5mol/L.

Still optionally further, the concentration of above-mentioned LiBr solution selects 9.3mol/L.

Further, will fibroin it is dry after be dissolved in dissolution in LiBr solution be will fibroin it is dry after be dissolved in LiBr solution, 0.8-1.2h is dissolved at 55-65 DEG C.

Still optionally further, will fibroin it is dry after be dissolved in dissolution in LiBr solution be will fibroin it is dry after be dissolved in LiBr solution In, 1h is dissolved at 60 DEG C.

S2, preparation silk fibroin bracket.

Silk fibroin bracket is made by the way that silk fibroin solution to be freeze-dried.

Further, the step of silk fibroin solution being freeze-dried, comprising:

After silk fibroin solution is mixed with glycerol, it is freeze-dried in -19~-21 DEG C, then again freezes first time for the first time Dry first fibroin bracket obtained is soaked in water, after removing glycerol, is carried out second and is freeze-dried, fibroin albumen branch is made Frame.

Further, silk fibroin solution is freeze-dried, comprising:

It after silk fibroin solution is mixed with glycerol, is freeze-dried in -20 DEG C, then again makes first time freeze-drying for the first time The the first fibroin bracket obtained is soaked in water, after removing glycerol, is carried out second and is freeze-dried, obtained silk fibroin bracket.

Illustratively, freeze-drying for the first time is the glycerol for being 60% by the silk fibroin solution of 2w/v% and its dry weight percentage It is uniformly mixed, pours into 96 orifice plates.12h is placed at -20 DEG C, is then freeze-dried.Second of freeze-drying is will be by first The fibroin bracket that secondary freeze-drying obtains is soaked in 48h in ultrapure water, and, to remove glycerol, every 2h replaces a water, freezes again dry Silk fibroin bracket is obtained after dry.

S3, the SBF solution immersed with silk fibroin bracket is irradiated using microwave, carries out mineralising reaction.

Still optionally further, the concentration of SBF solution is 1 × SBF solution.

When the progress mineralising to silk fibroin bracket common at present, SBF of the concentration of SBF solution usually at 2-10 times is molten Liquid, and it is only necessary to 1 times for fibroin albumen/hydroxyapatite compound rest preparation method that the application embodiment provides SBF solution, therefore it can be effectively reduced the dosage of SBF solution, it reduces and is polluted caused by residue SBF solution.

Optionally, above-mentioned 1 × SBF solution includes: 1.0MNaOH 40ml；HEPES 21.183g；NaCl 5.404g； KCl 0.225g；K₂HPO₄·3H₂O 0.231g；MgCl₂·3H₂O 0.311g；CaCl₂0.292g；Na₂SO₄0.072g； NaHCO₃0.355g and 1.0MNaOH 4.8mL.

It should be noted that being to sequentially add 1.0MNaOH 40ml in sequence when configuring above-mentioned 1 × SBF solution； HEPES 21.183g；NaCl 5.404g；KCl 0.225g；K₂HPO₄·3H₂O 0.231g；MgCl₂·3H₂O 0.311g； CaCl₂0.292g；Na₂SO₄0.072g；NaHCO₃0.355g and 1.0MNaOH 4.8mL.

It further, is in a sealed meter environment, to leaching to the SBF solution irradiation immersed with silk fibroin bracket using microwave Bubble has the SBF solution of silk fibroin bracket to carry out microwave irradiation.

Illustratively, being to the SBF solution irradiation immersed with silk fibroin bracket using microwave will be immersed with fibroin albumen The SBF solution of bracket, which is placed in micro-wave oven, carries out microwave irradiation.

Still optionally further, the power selection of micro-wave oven is 600-800W.

Illustratively, above-mentioned micro-wave oven carries out voltage 220V, microwave oven output power 700W when microwave irradiation.Microwave master Frequency is 2455MHz.

Further, use the time of microwave irradiation for 14-16S, the number of microwave irradiation is 8-10 times.

Still optionally further, use the time of microwave irradiation for 15S, the number of microwave irradiation is 9 times.

Within the time of above-mentioned microwave irradiation and the numbers range of microwave irradiation, it can guarantee hydroxyapatite pattern And ingredient.More than above-mentioned range, time too long solution temperature heating is excessively high, influence mineralising hydroxyapatite pattern and at Point, time too short reaction time is inadequate, and deposition effect is poor.

Illustratively, it will be placed in micro-wave oven immersed with the SBF solution of silk fibroin bracket and carry out microwave irradiation, opened micro- Wave furnace, microwave irradiation 15S are then shut off micro-wave oven 15S.Then micro-wave oven is opened again, and microwave irradiation 15S is then shut off microwave Furnace 15S；So circulation 9 times.

Still optionally further, it when being irradiated using microwave, can choose holding solution left standstill, carry out microwave irradiation, Can choose makes solution rotating, carries out microwave irradiation.

This fibroin albumen/hydroxyapatite compound rest preparation method that the application embodiment provides operates letter Single, the time is short, and mineralization velocity is fast, and high-efficient, the uniformity of mineralising is good.

In applying for some embodiments, it can choose and be repeated once above-mentioned step S3, it is molten to change a SBF halfway Liquid.

Embodiment further provides a kind of fibroin albumen/hydroxyapatite compound rests by the application some, using as above The fibroin albumen stated/hydroxyapatite compound rest preparation method is made.The fibroin albumen/hydroxyapatite compound rest Surface and internal all uniform depositions have hydroxyapatite.

The feature of the application and performance are described in further detail with reference to embodiments: