CN109248341B - Soft tissue repair patch and preparation method thereof - Google Patents

Abstract

The invention discloses a soft tissue repair patch and a preparation method thereof, wherein the repair patch comprises the following components: as a substrate of extracellular matrix and a natural protein outer coating, the surface of the coating is modified on the extracellular matrix through a layer-by-layer self-assembly technology to form the coating; the extracellular matrix is porcine small intestine submucosa; the natural protein is silk fibroin, and the silk fibroin is derived from silkworm cocoons. The composite acellular matrix material with the functionalized silk fibroin surface is prepared by a layer-by-layer self-assembly technology, and has the performance of enhancing the mechanical property and the degradation performance.

Description

Soft tissue repair patch and preparation method thereof

Technical Field

Aiming at the problem of mechanical retentivity of the conventional clinical soft tissue repair biological patch, the invention relates to a mechanical enhancement technology of a porcine small intestine submucosa acellular biological patch, which is a surface modification technology based on layer-by-layer self-assembly of silk fibroin protein.

Background

At present, the acellular biological patch based on animal tissue sources can inducibly repair various soft tissue defects, remarkably reduce the incidence rate of postoperative infection, foreign body reaction and tissue adhesion, greatly reduce the probability and severity of clinical adverse reaction, and have attracted great attention to the potential treatment application value. Therefore, the development of novel bioactive materials inducing tissue regeneration has become the development direction of the current biomedical materials.

The porcine Small Intestinal Submucosa (SIS) is a natural biological material rich in active factors, mainly comprises collagen, fibrin, elastin, a plurality of growth factors and signal molecules, has excellent biological activity, and can obtain complete reconstruction of tissue structure and function when being applied to repair of various tissue defects with specific and prominent biological advantages. In more than ten years, the SIS material is adopted abroad to treat various clinical diseases, over millions of patients are cured, and good clinical effects are obtained. However, under the action of various factors such as in vivo mechanics and enzymes, SIS is degraded too fast, so that the mechanical integrity of the SIS is lost, and the SIS is not beneficial to the remodeling of tissues.

Although a small amount of SIS-based biological patch products and functional dressing products are sold in the market in the domestic market, the clinical curative effect of the biological patch products and the functional dressing products needs to be improved, the clinical application range needs to be enlarged, the material design needs to be further optimized, the production process technology needs to be improved, and the material evaluation system is perfected. Currently, there are limitations associated with SIS-based biological patches, including reduced mechanical strength in an in vivo environment, reduced mechanical retention, and excessively fast degradation rates. According to the clinical application and the industrialization requirements of the biological patch and the functional dressing, the mechanical strength and the function of controllable degradation rate of the patch are improved by establishing a preparation technology of the natural protein surface functional patch.

The selected natural protein is silk fibroin approved by FDA, the silk fibroin has been clinically used as an operation suture line for many years, and compared with other natural polymers, the silk fibroin has excellent mechanical property, good biocompatibility, controllable degradation and absorbability, easy processing and wide sources. The surface modification technology adopts a layer-by-layer self-assembly technology of silk fibroin, and the unique amino acid sequence of the silk fibroin induces the formation of a beta-sheet secondary structure after the silk fibroin is processed, so that the connection between layers is realized.

Disclosure of Invention

The invention provides a novel acellular matrix composite material aiming at the problems and the defects of the existing soft tissue repair material. The composite material has good biological activity, mechanical property, controllable degradability, safety and no toxicity.

In order to achieve the technical purpose, the invention is specifically realized by the following technical scheme:

a soft tissue repair patch comprising:

as a substrate of extracellular matrix and a natural protein outer coating, the silk fibroin self-assembly surface is modified on the extracellular matrix layer by layer to form;

the extracellular matrix is porcine small intestine submucosa;

the natural protein outer coating is silk fibroin, and the silk fibroin is derived from silkworm cocoons.

The silk fibroin is prepared by the following method:

1) taking a certain amount of silk, and cutting into pieces;

2) preheating 3L of distilled water to 100 deg.C, adding 0.02M Na₂CO₃；

3) Slowly adding silk and Na₂CO₃Boiling the aqueous solution for 1 h;

4) pouring out the silk, and washing with cold distilled water;

5) repeating (2) - (4);

6) preheating 3L of distilled water to 100 deg.C, and boiling the silk in boiling water for 10 min;

7) washing degummed silk (fibroin) obtained in the above steps with cold water, drying in a fume hood, and weighing;

8) dissolving the fibroin obtained in the step (7) in a prepared LiBr solution, stirring until the fibroin is completely dissolved to obtain a fibroin solution, and placing the solution at 65 ℃ for 2 h;

9) filtering the silk fibroin solution obtained in the step (8) by using a 0.45-micron membrane;

10) and dialyzing with ultrapure water to obtain the silk fibroin solution required by the experiment.

In another aspect of the invention, a preparation method of the dura mater repair patch is provided, which comprises the following steps:

1) cutting the extracellular matrix into a square shape, and placing the square extracellular matrix into a 12-hole plate;

2) adding silk fibroin, and shaking on ice for 10min to make it uniformly adhere;

3) sucking out silk fibroin, adding ultrapure water, shaking on ice for 10min, and repeating twice;

4) sucking out ultrapure water, adding methanol, shaking on ice for 10min, sucking out methanol, and drying with nitrogen;

5) and repeating the steps until the soft tissue repair patch with the required number of layers is obtained.

The invention has the beneficial effects that:

1) the mechanical property is excellent. The composite material after coating has excellent mechanical property due to the self-grading structure of the silk fibroin, so that the material after being coated with the silk fibroin has integrally reinforced mechanical property, and after being treated by methanol, the beta-sheet structure in the silk fibroin is formed to play a role in physical crosslinking and also play a role in reinforcing the integral mechanical property.

2) The biological activity and the biocompatibility are better. The components in the composite material are all composed of natural components. The base SIS is an extracellular matrix component, the main component is collagen and contains various growth factors and active factors, and the outer coating silk fibroin is a natural protein.

3) Preventing rapid degradation. The silk fibroin amino acid sequence of the composite material outer coating contains a hydrophobic region with a high glycine repetitive sequence, and after methanol treatment, the hydrophobic sequence is exposed outside, so that the overall hydrophobicity of the material is increased, and the degradation rate is reduced.

4) The degradation rate is controllable. The degradation of the biomaterial should be at a controlled rate to meet the requirement for new tissue generation. Degradation of the composite material after the silk protein coating can be controlled by the contents of water-insoluble silk II and water-soluble silk I, and the content of the silk II can be increased after the composite material is treated by methanol, wherein the silk II contains a beta-sheet structure, and the degradation time of the silk can be increased due to the generation of the beta-sheet structure; this beta-sheet structure, in turn, can be reduced by heating or slow drying, thereby reducing the degradation time of the silk.

5) Is safe and nontoxic. The composite material is a material with natural sources, and the degradation product is safe and non-toxic, and no toxic reagent is adopted in the manufacturing process. Although treated with methanol, the methanol quickly volatilized after treatment with nitrogen gas and remained almost no.

6) The manufacturing process is simple and the cost is low. The composite material has simple preparation process, convenient production, wide silk source for the coating and low cost.

Drawings

FIG. 1 is a stress-strain curve for different layers of dural repair patches of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Extracting silk fibroin, comprising the following steps:

1) taking a certain amount of silk, and cutting into pieces;

2) preheating 3L of distilled water to 100 deg.C, adding 0.02M Na₂CO₃6.36g；

3) Slowly adding silk and Na₂CO₃Boiling the aqueous solution for 1 h;

4) pouring out the silk, and washing with cold distilled water;

5) repeating (2) - (4);

6) preheating 3L of distilled water to 100 deg.C, and boiling the silk in boiling water for 10 min;

7) washing the degummed silk (fibroin) obtained in the step with cold water, drying in a fume hood, and weighing;

8) preparing a LiBr solution: v_LiBr＝W_{Silk fibroin}*5

W_LiBr＝11mol/L*V_LiBr*86.85g/mol

Dissolving the fibroin obtained in the step (7) in the prepared LiBr solution at room temperature, stirring until the fibroin is completely dissolved to obtain a fibroin protein solution, and placing the solution at 65 ℃ for 2 hours;

9) filtering the silk fibroin solution obtained in the step (8) by using a 0.45-micron membrane;

10) dialyzing with ultrapure water to obtain the silk fibroin solution required by the experiment.

The method for preparing the dura mater repair patch by utilizing a layer-by-layer self-assembly method comprises the following steps:

1) the SIS is cut into a square with the size of 1.5 multiplied by 1.5cm and is put into a 12-hole plate, and one plate is put in each hole;

2) adding 1mg/ml silk protein, and shaking on ice for 10 min;

3) sucking out the silk, adding ultrapure water, and shaking on ice for 1min twice;

4) sucking out ultrapure water, adding 90% methanol, and shaking on ice for 10 min;

5) sucking out methanol, and drying by nitrogen;

6) repeating the steps (2) to (5) to the required number of layers;

example 2

Cutting original SIS and SIS coated with 5 and 10 layers of silk fibroin into 3mm × 30 mm; the tensile test is carried out in an MTS universal tensile testing machine with a 200N sensor, and the test temperature is normal temperature.

Measuring the thickness of a sample, fixing the sample on a clamp of a tensile testing machine, and recording the distance between the two clamps as L; setting test operation parameters, and setting the operation speed to be 1 mm/min; the tensile experiment was started and the graph abscissa and ordinate were set, the Y-axis data being force and the X-axis data being displacement.

And (3) converting the force-displacement curve and the distance between the material size and the clamp into a stress-strain curve:

the stress is force/cross-sectional area of the sample is thickness x width;

strain is Δ L/L, Δ L is the varying displacement, and L is the original inter-clamp distance.

The result is shown in figure 1, and the mechanical property of the whole material is enhanced along with the increase of the silk fibroin coating. The concrete characteristics are that the tensile strength of the material is increased, namely the borne tension is increased, the breaking elongation of the material is increased, namely the toughness of the material is increased, and the deformation resistance is enhanced.

Example 3

Cutting the SIS into the size of 1cm by a blade, coating the SIS with the size with silk fibroin, wherein the number of the original SIS and the number of the coated SIS are respectively 24, the number of the coated SIS is 12, the experiment is divided into a control group and an experiment group, the control group is two groups of the original SIS and the SIS coated with 5 layers of silk fibroin, and the degradation solution is PBS; the experimental group is three groups of original SIS, SIS coated with 5 layers of fibroin and SIS coated with 10 layers of fibroin, and the degradation solution is PBS solution containing 1u/ml collagenase (type I); each set was 4 replicates.

Putting the samples into 15ml centrifuge tubes, marking one sample in each tube, putting all the test tubes into a 37-DEG constant temperature incubator, changing the liquid every 24h, setting the sampling time to be three time points of 1 day, 3 days and 7 days, taking out the samples, taking pictures, and recording the states of the samples when each set time point is reached.

Results analysis the control group showed no significant change in sample size over the selected time. In the experimental group, when the original SIS group is degraded for one day, the original form is basically maintained, the original form is incomplete on the third day, and the original form is completely degraded on the 7 th day; the SIS group coated with 5 layers of fibroin has basically complete original shape on the first day and the third day, and the shape is changed on the 7 th day; the morphology of the samples was not significantly changed at each time point in the SIS group coated with 10 layers of fibroin. It can be seen that the degradation performance of the SIS material modified by silk fibroin is enhanced, and the degradation prevention performance is different after the SIS material is modified by silk fibroin with different layers.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (2)

1. A soft tissue repair patch, comprising:

as a substrate of extracellular matrix and a natural protein outer coating, natural protein is coated on the extracellular matrix through layer-by-layer self-assembly to form a film;

the extracellular matrix is porcine small intestine submucosa;

the natural protein outer coating is silk fibroin, and the silk fibroin is derived from silkworm cocoons;

the preparation method of the soft tissue repair patch comprises the following steps:

1) cutting the extracellular matrix into a square shape, and placing the square extracellular matrix into a 12-hole plate;

2) adding silk fibroin, and shaking on ice for 10min to make it uniformly adhere;

3) sucking out silk fibroin, adding ultrapure water, shaking on ice for 1min, and repeating twice;

4) sucking out ultrapure water, adding methanol, shaking on ice for 10min, sucking out methanol, and drying with nitrogen;

5) and repeating the steps until the soft tissue repair patch with the required number of layers is obtained.

2. The soft tissue repair patch according to claim 1, wherein the silk fibroin is prepared by the following method:

1) taking a certain amount of silk, and cutting into pieces;

2) preheating 3L of distilled water to 100 deg.C, adding 0.02M Na₂CO₃；

3) Slowly adding silk and Na₂CO₃Boiling the aqueous solution for 1 h;

4) pouring out the silk, and washing with cold distilled water;

5) repeating steps (2) - (4);

6) preheating 3L of distilled water to 100 deg.C, and boiling the silk in boiling water for 10 min;

7) washing the degummed fibroin obtained in the step with cold water, drying in a fume hood, and weighing;

8) dissolving the fibroin obtained in the step (7) in a prepared LiBr solution, stirring until the fibroin is completely dissolved to obtain a fibroin solution, and placing the solution at 65 ℃ for 2 h;

9) filtering the silk fibroin solution obtained in the step (8) by using a 0.45-micron membrane;

10) and dialyzing with ultrapure water to obtain the silk fibroin solution required by the experiment.

Images