CN112972766B - High mechanical strength silk fibroin-hydroxyapatite composite bone scaffold and preparation method thereof - Google Patents

Abstract

The invention relates to a silk fibroin-hydroxyapatite composite bone scaffold with high mechanical strength and a preparation method thereof. The silk fibroin solution and the hydroxyapatite aqueous dispersion are subjected to ultrasonic action to form a mixed solution, and the silk fibroin/hydroxyapatite composite three-dimensional porous scaffold with high mechanical strength is rapidly and nontoxically prepared by a direct current directional electric field induction method.

Description

High mechanical strength silk fibroin-hydroxyapatite composite bone scaffold and preparation method thereof

technical field

The invention relates to the technical field of medical materials, in particular to a high mechanical strength silk fibroin-hydroxyapatite composite bone support and a preparation method thereof.

Background technique

Bone is the second most commonly transplanted tissue in the world. At least 4 million operations are performed every year. Bone defects will bring great inconvenience to people's body and life. Although bone has a certain regenerative ability, in most cases, it is still necessary to improve the bone repair ability. In clinical practice, autologous bone transplantation or allogeneic bone is commonly used in the treatment of bone defects, but there are problems such as immune resistance and limited sources. In view of this, people are committed to researching bone tissue engineering scaffold materials that can mimic the structure and performance of natural bone. In recent years, people have paid more and more attention to natural polymers due to its better biocompatibility and multifunctionality. This advantageous natural polymer has been proven to have certain bone repair properties.

Silk fibroin is a natural polymer material derived from silk, and its research and application in the field of bone tissue engineering are increasing. At present, compared with natural bone, the mechanical properties and osteogenic properties of porous scaffolds prepared by regenerated silk fibroin are poor. In order to improve this shortcoming, hydroxyapatite particles can be added to more effectively simulate the natural bone structure. Hydroxyapatite is a bioactive ceramic whose chemical composition and crystal structure are similar to the main components of inorganic substances in human bone matrix. Previous studies have shown that artificially synthesized hydroxyapatite has the same composition and similar structure as hydroxyapatite in human bones, has no adverse reactions when implanted in the human body, has good biocompatibility, and exhibits a certain degree of osteogenesis Inductive and osteoconductive. Therefore, using silk fibroin as a matrix and incorporating hydroxyapatite particles, a scaffold with excellent mechanical properties and bone repair performance can be prepared.

At present, there are two main methods for the preparation of silk fibroin/hydroxyapatite composite bone scaffolds. One is to directly mix silk fibroin and hydroxyapatite mechanically, and then prepare composite scaffolds by methods such as particle filtration, foaming, and direct freeze-drying. Bone scaffold, the second is to prepare a pure silk fibroin scaffold, and introduce hydroxyapatite through a biomimetic mineralization method. These preparation methods have many operation steps, and the mechanical properties of the obtained composite bone scaffolds are relatively poor, which is unfavorable for clinical use.

Contents of the invention

In order to solve the above-mentioned technical problems, the present invention rapidly and non-toxicly prepares silk fibroin/hydroxyapatite composite bone scaffold through the method of direct current directional electric field induction, the preparation cycle is short and the operation is simple, and the final scaffold has excellent and adjustable mechanical properties. performance.

The first object of the present invention is to provide a method for preparing a high mechanical strength silk fibroin-hydroxyapatite composite bone scaffold, comprising the following steps:

S1. Disperse hydroxyapatite in water by ultrasonic action, then add silk fibroin solution to the uniformly dispersed hydroxyapatite aqueous solution according to the mass ratio of silk fibroin to hydroxyapatite 10:0.5~5, and mix by ultrasonic Uniformly, obtain silk fibroin/hydroxyapatite solution;

S2. Insert two graphite plates into the silk fibroin/hydroxyapatite solution, and connect the positive and negative poles of the DC power supply respectively, and perform directional electric field induction on the silk fibroin/hydroxyapatite solution, so that the silk fibroin solution will automatically The assembled nano-particles are aggregated into micro-particles near the positive plate, and hydroxyapatite is wrapped inside to obtain a silk fibroin/hydroxyapatite composite gel;

S3. Pre-cooling the silk fibroin/hydroxyapatite composite gel and then freeze-drying to obtain the silk fibroin-hydroxyapatite composite bone scaffold.

Further, the directional electric field induction is treated at a voltage of 20-30V for 10-40 minutes.

Further, the concentration of the silk fibroin solution is 5-10% by weight.

Further, the silk fibroin solution is prepared by the following method: degumming silkworm cocoons, washing and drying after degumming, dissolving in LiBr solution after drying, filtering, dialysis and centrifugation to obtain silk fibroin solution.

Further, the degumming treatment is to put silkworm cocoons in 0.1-1% w/v NaHCO ₃ solution and boil for 30-60 minutes.

Further, the concentration of the LiBr solution is 9-10 mol/L.

Further, the pre-cooling is placed at -10 to -30°C for 10 to 20 hours.

Further, the freeze-drying is carried out by freezing at -80°C for 1-5 hours and then using a freeze-drying machine.

The second object of the present invention is to provide a silk fibroin-hydroxyapatite composite bone scaffold prepared by the method.

The third object of the present invention is to provide the application of the silk fibroin-hydroxyapatite composite bone scaffold in preparing artificial bones.

By means of the above solution, the present invention has at least the following advantages:

The method of the invention has simple operation, short preparation period, environmental protection, safety and no pollution, and the prepared silk fibroin/hydroxyapatite composite bone scaffold has excellent mechanical properties and can meet the requirements of clinical use.

The above description is only an overview of the technical solutions of the present invention. In order to understand the technical means of the present invention more clearly and implement them according to the contents of the description, the preferred embodiments of the present invention are described below with detailed drawings.

Description of drawings

Fig. 1 is the schematic diagram of preparing silk fibroin/hydroxyapatite composite bone scaffold under the action of electric field;

Fig. 2 is the electron microscope picture of two kinds of composite bone scaffolds;

Fig. 3 is the infrared characterization diagram of two kinds of composite bone scaffolds;

Fig. 4 is the XRD characterization figure of two kinds of composite bone scaffolds;

Fig. 5 is the comparison diagram of the mechanical properties of two kinds of composite bone scaffolds;

Fig. 6 is a comparison diagram of the mechanical properties of the electric field composite bone scaffold and cancellous bone and the silk fibroin/hydroxyapatite composite bone scaffold in the reference.

Detailed ways

Below in conjunction with the examples, the specific implementation of the present invention will be further described in detail. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Composite bone scaffold for compression mechanical properties test method:

A polytetrafluoroethylene mold was used to prepare the scaffold into a cylindrical shape with a size of 10 mm (diameter) × 8 mm (height), and a texture analyzer (TMS-PRO) was used to test the compressive mechanical properties of the sample. The test speed is set at 10mm/min, the trigger force is 0.03N, the compression ratio is 80%, and three samples are tested in each group.

Example 1:

According to the schematic diagram in Figure 1, prepare the composite bone scaffold:

1. Cut the cocoon into cocoon pieces of about ^1cm2 , weigh a certain amount of cocoon cocoon pieces, put them into the NaHCO ₃ solution containing 0.5% w/v, boil for 45min, and use warm deionized water to degummed silk Rinse 4-5 times, and after drying, dissolve silk fibroin in 9.3 mol/L LiBr solution and dissolve at 60°C for 1 hour. After filtration, dialysis and centrifugation, a silk fibroin solution with a final concentration of about 9wt% was obtained.

2. According to the mass fraction of silk fibroin: hydroxyapatite = 10:2, the hydroxyapatite is first dispersed in a certain amount of water, then the silk fibroin is added, and the two are mixed evenly by ultrasonication.

3. Under the voltage of 25V, conduct direct current directional electric field induction treatment of silk fibroin/hydroxyapatite solution (about 20-30min).

4. Take out the silk fibroin/hydroxyapatite composite gel, put it at -20°C overnight, then freeze it at -80°C for 4 hours, and put it into a freeze dryer to freeze dry.

5. Take out the freeze-dried scaffold to obtain a composite bone scaffold.

Example 2:

1. Cut the cocoon into cocoon pieces of about ^1cm2 , weigh a certain amount of cocoon cocoon pieces, put them into the NaHCO ₃ solution containing 0.5% w/v, boil for 45min, and use warm deionized water to degummed silk Rinse 4-5 times, and after drying, dissolve silk fibroin in 9.3 mol/L LiBr solution and dissolve at 60°C for 1 hour. After filtration, dialysis and centrifugation, a silk fibroin solution with a final concentration of about 9wt% was obtained.

2. According to the mass fraction of silk fibroin: hydroxyapatite = 10:1, the hydroxyapatite is first dispersed in a certain amount of water, then the silk fibroin is added, and the two are mixed evenly by ultrasonication.

3. Under the voltage of 20V, conduct direct current directional electric field induction treatment of silk fibroin/hydroxyapatite solution (about 30-40min).

4. Take out the silk fibroin/hydroxyapatite composite gel, put it at -20°C overnight, then freeze it at -80°C for 4 hours, and put it into a freeze dryer to freeze dry.

5. Take out the freeze-dried scaffold to obtain a composite bone scaffold.

Example 3:

1. Cut the cocoon into cocoon pieces of about ^1cm2 , weigh a certain amount of cocoon cocoon pieces, put them into the NaHCO ₃ solution containing 0.5% w/v, boil for 45min, and use warm deionized water to degummed silk Rinse 4-5 times, and after drying, dissolve silk fibroin in 9.3 mol/L LiBr solution and dissolve at 60°C for 1 hour. After filtration, dialysis and centrifugation, a silk fibroin solution with a final concentration of about 9wt% was obtained.

2. According to the ratio of mass fraction silk fibroin: hydroxyapatite = 10:3, the hydroxyapatite is first dispersed in a certain amount of water, then the silk fibroin is added, and the two are mixed evenly through ultrasonication.

3. Under the voltage of 30V, conduct direct current directional electric field induction treatment of silk fibroin/hydroxyapatite solution (about 30-40min).

4. Take out the silk fibroin/hydroxyapatite composite gel, put it at -20°C overnight, then freeze it at -80°C for 4 hours, and put it into a freeze dryer to freeze dry.

5. Take out the freeze-dried scaffold to obtain a composite bone scaffold.

Comparative example 1:

1. Cut the cocoon into cocoon pieces of about ^1cm2 , weigh a certain amount of cocoon cocoon pieces, put them into the NaHCO ₃ solution containing 0.5% w/v, boil for 45min, and use warm deionized water to degummed silk Rinse 4-5 times, and after drying, dissolve silk fibroin in 9.3 mol/L LiBr solution and dissolve at 60°C for 1 hour. After filtration, dialysis and centrifugation, a silk fibroin solution with a final concentration of about 9wt% was obtained.

2. According to the ratio of mass fraction silk fibroin: hydroxyapatite = 10:2, first disperse the hydroxyapatite in a certain butanol solution, mix well by ultrasonic, add dropwise into the silk fibroin solution, and add While mixing the solution.

3. Put the mixed solution at -20°C overnight, then freeze at -80°C for 4 hours, and put it into a freeze dryer to freeze dry.

4. Take out the freeze-dried scaffold to obtain the composite bone scaffold.

The performance of the composite bone scaffold obtained by comparative example 1 and comparative example 1, the results are as follows:

Figure 2 is the electron micrographs of two kinds of composite bone scaffolds, and the results show that both composite bone scaffolds exhibit porous structures, and the pore diameter of the butanol composite bone scaffolds is relatively uniform, with a size of 50-100 μm. The pore size of the electric field composite bone scaffold is mostly concentrated in 0-50 μm, and some large pores of 200-300 μm appear at the same time, showing a multi-level porous structure.

Fig. 3 is the infrared characterization figure of two kinds of composite bone scaffolds, the result shows: the FTIR absorption peak of butanol composite bone scaffold material amide I mainly appears at ^1621cm Folding is the main factor, while the FTIR absorption peak of amide I of the electric field composite bone scaffold material mainly appears at 1637cm ^-1 , indicating that the silk fibroin in the electric field composite bone scaffold material is dominated by random coils. Both composite bone scaffolds have absorption peaks at 1030cm ^-1 , 600cm ^-1 , and 560cm ^-1 , which correspond to the PO ₄ ^3- group in hydroxyapatite, indicating that hydroxyapatite is successfully compounded on silk fibroin in the protein scaffold.

Figure 4 shows the XRD characterization diagrams of two kinds of composite bone scaffolds. The results show that the composite bone scaffolds prepared by two different methods all have characteristic peaks at 26°, 31.7°, 33°, and 34°, corresponding to hydroxyapatite respectively. (002), (211), (300), (202) crystal planes. Both the results and IR showed that the silk fibroin/hydroxyapatite composite bone scaffolds were successfully prepared by both methods.

Figure 5 is a comparison chart of the mechanical properties of the two composite bone scaffolds, and the results show that: compared with the butanol composite bone scaffold, the compressive strength of the electric field composite bone scaffold is greatly improved.

Table 1 shows the maximum compressive strength and elastic modulus of the two composite bone scaffolds. The results show that compared with the butanol composite bone scaffolds, the maximum compressive strength and elastic modulus of the electric field composite bone scaffolds have been greatly improved, respectively reaching butanol composite bone scaffolds. 14 times and 21 times that of alcohol composite bone scaffold.

Table 1

sample Maximum compression strength (MPa) Elastic modulus (MPa) Butanol Composite Bone Scaffold 1.75±0.18 1.35±0.17 Electric Field Composite Bone Scaffold 24.66±0.88 28.91±3.19

Figure 6 is a comparison of the mechanical properties of the electric field composite bone scaffold with cancellous bone and some silk fibroin/hydroxyapatite composite bone scaffolds in the references. The results show that the mechanical properties of the electric field composite bone scaffold are better than most of the references. It has been greatly improved, and meets the mechanical properties requirements of natural cancellous bone, which can meet the clinical application, and is an effective new strategy.

The above are only preferred embodiments of the present invention, and are not intended to limit the present invention. It should be pointed out that for those of ordinary skill in the art, some improvements and modifications can be made without departing from the technical principles of the present invention. , these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims (5)

1. a preparation method of high mechanical strength silk fibroin-hydroxyapatite composite bone scaffold, is characterized in that, comprises the steps: S1. Disperse hydroxyapatite in water by ultrasonic action, then add silk fibroin solution to the uniformly dispersed hydroxyapatite aqueous solution according to the mass ratio of silk fibroin to hydroxyapatite 10:0.5~5, and mix by ultrasonic Uniformly, obtain silk fibroin/hydroxyapatite solution; S2. Insert two graphite plates into the silk fibroin/hydroxyapatite solution, and connect the positive and negative poles of the DC power supply respectively, and perform directional electric field induction on the silk fibroin/hydroxyapatite solution, so that the silk fibroin solution will automatically The nanoparticles formed by the assembly are aggregated into micron particles near the positive plate, and hydroxyapatite is wrapped inside to obtain a silk fibroin/hydroxyapatite composite gel; S3. Pre-cooling the silk fibroin/hydroxyapatite composite gel and then freeze-drying to obtain the silk fibroin-hydroxyapatite composite bone scaffold; The directional electric field induction effect is treated at a DC stable voltage of 20-30V for 10-40min; the concentration of the silk fibroin solution is 5-10wt%; silkworm cocoons are degummed, washed with water after degumming, and dried. Dissolve in LiBr solution, obtain silk fibroin solution after filtration, dialysis, and centrifugation; the degumming treatment is to place silkworm cocoons in 0.1-1% w/v NaHCO ₃ solution, and boil for 30-60 minutes; The concentration of the LiBr solution is 9-10 mol/L. 2. The method according to claim 1, characterized in that the pre-cooling is placed at -10 to -30°C for 10 to 20 hours. 3. The method according to claim 1, characterized in that the freeze-drying is carried out by using a freeze dryer after freezing at -80°C for 1-5 hours. 4. A silk fibroin-hydroxyapatite composite bone scaffold prepared by the method according to any one of claims 1 to 3. 5. The application of the silk fibroin-hydroxyapatite composite bone scaffold according to claim 4 in the preparation of artificial bones.

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