CN110237304A - A kind of multi-level structure support and preparation method thereof - Google Patents

Abstract

The present invention relates to a kind of multilevel structure brackets and preparation method thereof, comprising the following steps: step 1 obtains natural source extracellular matrix；Step 2, the first spinning solution of preparation and the second spinning solution；Step 3, the first composite nano fibre yarn line of preparation；Step 4, preparation are in the mechanics enhancement layer of planar structure；Step 5, preparation are in the form of a column the substrate holder of structure；Step 6 makes to prepare multilevel structure bracket.It the advantage is that, use natural source extracellular matrix extract as electrospun material, sticking, be proliferated and migrating with good facilitation to cell be conducive to host cell and quickly cover with tough belt supporting frame, realize rapid regeneration；High molecular material can play the role of supporting newborn ligament tissue after the degradation of natural source extracellular matrix extract；The inside and outside of multilevel structure bracket is coated with composite nanometer particle gel, can guide, promote the fast-growth of osteocyte, to quickly rebuild tendon-bone.

Description

A kind of multi-level structure support and preparation method thereof

technical field

The invention relates to the technical field of repair brackets, in particular to a multi-level structure bracket and a preparation method thereof.

Background technique

The knee joint is the joint with the largest amount of motion and the most complex structure in the whole body. Its structural and mechanical characteristics make it prone to injury. Anterior Cruciate Ligaments (ACL) is one of the most common damaged parts. According to statistics, there are about 300,000 ACL reconstruction operations per year in the United States, and a conservative estimate of at least 600,000 ACL injuries per year in my country. Equipment is in great demand. After ACL injury, it lacks self-regeneration and repair ability, so autologous tendon transplantation is still the first choice for clinical treatment today, but there are limitations such as limited source of graft and damaged donor site.

In the existing treatment methods, allogeneic tendons and artificial synthetic ligaments are commonly used, but both of them have certain defects. Allogeneic tendons are difficult to meet clinical needs and have the risk of spreading infectious diseases; artificial synthetic ligaments have defects such as inability to induce ligament regeneration, failure to achieve physiological tendon-bone healing, and easy failure. Therefore, there is an urgent clinical need for artificial ligament implantation devices that can induce ligament regeneration.

Natural source extracellular matrix extract is a tissue regeneration inducing material widely used in tissue engineering scaffolds. Its active ingredients are collagen and elastin, which are natural material components of the human body. Although the nanofiber scaffold prepared by electrospinning technology has certain mechanical properties, due to the small pores, the host cells cannot normally migrate and grow into the nanofiber, and it is difficult to achieve rapid reconstruction of new tissue only through physical adhesion. , which requires the combination of weaving technology and thermal phase separation technology combined with electrospinning technology to functionalize the ligament scaffold in modules, so that it has a multi-layer structure, and each performs its duties to meet the requirements of clinical transplantation.

Electrospinning technology is widely used in the preparation of tissue engineering scaffolds, and the application of weaving technology in the preparation of ligament repair scaffolds has also been reported in the literature. Since the thermally induced phase separation technology is limited in the choice of materials for the preparation of nanofibers, although it is not as widely used as the former two, it is also very good in the field of scaffolds prepared by blending with other materials. However, so far, there have been no reports on the preparation of ligament repair scaffolds by combining electrospinning technology, weaving technology and thermal phase separation technology.

Therefore, there is an urgent need for a scaffold for ligament repair prepared by electrospinning technology, braiding technology and thermal phase separation technology, which has the advantages of integrating mechanical properties and biocompatibility.

Contents of the invention

The object of the present invention is to provide a multi-level structure support and a preparation method thereof in view of the deficiencies in the prior art.

One aspect of the present invention is to provide a method for preparing a multi-level structure support, comprising the following steps:

Step 1. Obtain natural source extracellular matrix, freeze-dry and store at 4°C to -80°C;

Step 2, using a solvent to dissolve the natural source extracellular matrix and the polymer material to obtain a clarified first spinning solution and a second spinning solution;

Step 3, using the first spinning solution to perform electrospinning to prepare the first composite nanofiber yarn;

Step 4, using the first composite nanofiber yarn to prepare a mechanical reinforcement layer with a planar structure;

Step 5, coating composite nanoparticle gel on the front and back of the mechanically enhanced layer to form a gel layer, and then performing wrapping and thermally induced phase separation to obtain a first scaffold with a columnar structure;

Step 6. Electrospinning is performed using the second spinning solution, and a layer of the second composite nanofiber is covered on the outer surface of the first scaffold to form a second scaffold, so as to prepare a multi-level scaffold.

Preferably, the content of each component of the first spinning solution and the second spinning solution is the same or different.

Preferably, in step 1, the method for obtaining the natural source extracellular matrix is:

Washing, crushing, degumming, dialysis, and freeze-drying of pigtails or rat tails or silkworm chrysalis in sequence, and then spraying several times with phosphate buffer containing penicillin/streptomycin double antibodies to obtain the natural source extracellular matrix .

Preferably, the polymer material is poly-4-hydroxybutyrate, a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate, a copolymer of polyhydroxyalkanoate, polylactic acid and polycaprolactone Any one or a combination of polydioxanone, poly(lactic-co-glycolic acid) and biomedical degradable polyurethane.

Preferably, the solvent is hexafluoroisopropanol, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, dichloromethane, acetone, chloroform, trifluoroacetic acid, trifluoroethanol any one or a combination of several.

Preferably, in the step 2, the parts by weight of the components of the first spinning solution are as follows:

Natural source extracellular matrix extract 5-50 parts

Polymer material 50～95 parts

100 parts of solvent.

Preferably, in the step 2, the parts by weight of the components of the second spinning solution are as follows;

Natural source extracellular matrix extract 50-95 parts

Polymer material 5-50 parts

100 parts of solvent.

Preferably,

The composite nanoparticle gel is composed of the following components by weight:

Preferably, the therapeutic factor is any one or a combination of several of BMP-2, deferoxamine, KGN, VEGF, dexamethasone, eugenol, chondroitin sulfate, and interleukin.

Preferably, the water is ultrapure water.

Preferably, in the first composite nanofiber yarn, the nanofibers are inclined by 10°-80° along the axial direction of the first composite nanofiber yarn.

Preferably, in the step 2, the preparation method of the first spinning solution and the second spinning solution is:

Step 21. Weighing certain parts by weight of the natural extracellular matrix extract and the polymer material;

Step 22. Under the condition of 50°C-80°C, dissolve the natural source extracellular matrix and the polymer material in the solvent, and stir to dissolve, so as to obtain the clarified first spinning solution and the the second spinning solution;

Wherein, the mass volume ratio of the first spinning solution to the second spinning solution is 2%-30%.

Preferably, in the step 4, the first composite nanofiber yarn is prepared into the mechanical reinforcement layer by means of doubling, twisting and weaving.

Preferably, in the step 4, the first composite nanofiber yarn is prepared into the mechanical reinforcement layer by means of doubling, twisting and weaving.

Preferably, in the step 4, the first composite nanofiber is prepared into the mechanical reinforcement layer by means of doubling and twisting knitting.

Preferably, the first composite nanofiber yarn contains a reinforcing core layer or does not contain a reinforcing core layer.

Preferably, the reinforcing core layer is poly-4-hydroxybutyrate, a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate, a copolymer of polyhydroxyalkanoate, polylactic acid and polycaprolactone It is made of any one or a combination of polydioxanone, polylactic acid-glycolic acid copolymer, and biomedical degradable polyurethane.

Preferably, the preparation method of the reinforced core layer is:

Preparation of monofilaments with a diameter of 50 μm to 300 μm by melt drawing;

4-8 monofilaments are divided into one strand, and 2-10 strands are twisted to obtain a reinforced core layer.

Another aspect of the present invention is to provide a multi-level structure scaffold prepared by the above preparation method.

The present invention adopts the above technical scheme, and compared with the prior art, it has the following technical effects:

A multi-level structure scaffold and its preparation method of the present invention uses natural source extracellular matrix extract as the electrospinning material, which has a good promoting effect on cell adhesion, proliferation and migration, and is conducive to the rapid growth of host cells with ligaments scaffold to achieve rapid regeneration; polymer materials can support the new ligament tissue after the degradation of natural extracellular matrix extracts; the inside and outside of the multi-level structure scaffold are coated with composite nanoparticle gel, which can guide and promote bone regeneration Rapid growth of cells, thereby rapidly rebuilding tendon-bone.

Description of drawings

Fig. 1 is a schematic diagram of the preparation process of the multi-level structure scaffold of the present invention.

Figure 2a is a schematic diagram of a first composite nanofiber yarn of the present invention.

Figure 2b is a scanning electron microscope image of the first composite nanofiber yarn of the present invention.

Figure 3a is a schematic diagram of the composite nanoparticle gel of the present invention.

Fig. 3b is a transmission electron microscope image of the composite nanoparticle gel of the present invention.

Fig. 4a is the scanning electron micrograph of the hierarchical structure scaffold coated with composite nanoparticle gel of the present invention

Fig. 4b is a scanning electron microscope image of a section of the multi-level postganglionic scaffold coated with composite nanoparticle gel of the present invention.

Fig. 5 is a schematic diagram of a multi-level structural support according to an exemplary embodiment of the present invention.

Fig. 6 is a cross-sectional view of a multi-level structural support according to an exemplary embodiment of the present invention.

The reference signs therein are: the first support 100 ; the gel layer 200 ; the second support 300 .

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

It should be noted that, in the case of no conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but not as a limitation of the present invention.

Example 1

A schematic embodiment of the present invention, as shown in Figures 1 to 6, a method for preparing a multi-level structure scaffold, comprising the following steps:

Step 1. Obtain natural source extracellular matrix, freeze-dry and store at 4°C to -80°C;

Step 2, using a solvent to dissolve the natural source extracellular matrix and the polymer material to obtain a clarified first spinning solution and a second spinning solution;

Step 3, using the first spinning solution to perform trumpet-conjugated electrospinning to prepare the first composite nanofiber yarn;

Step 4, using the first composite nanofiber yarn to prepare a mechanical reinforcement layer with a planar structure;

Step 5. Coating composite nanoparticle gel on the front and back of the mechanically enhanced layer to form a gel layer 200, and then performing wrapping and thermal phase separation treatment to obtain the first columnar structure 100;

Step 6. Use the second spinning solution to perform trumpet-conjugated electrospinning, and cover the outer surface of the first scaffold 100 with a layer of second composite nanofibers to form the second scaffold 300 to prepare a multi-level scaffold.

Further, in step 1, the method for obtaining natural source extracellular matrix is:

Pig tail or rat tail or silkworm chrysalis are washed, pulverized, degummed, dialyzed, freeze-dried sequentially, and then sprayed with phosphate buffer containing penicillin/streptomycin double antibody several times to obtain natural extracellular matrix.

Among them, pigtails were taken from 3-month-old healthy animals, rat tails were taken from 4-week-old healthy animals, and silkworm chrysalis were tussah silkworm chrysalis.

Further, spray wash 10-20 times with phosphate buffer solution containing 0.5%-1% penicillin/streptomycin double antibody.

Further, the polymer material is poly-4-hydroxybutyrate, a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate, a copolymer of polyhydroxyalkanoate, polylactic acid and polycaprolactone, Any one or a combination of polydioxanone, poly(lactic-co-glycolic acid) and biomedical polyurethane.

Further, the solvent is hexafluoroisopropanol, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, dichloromethane, acetone, chloroform, trifluoroacetic acid, trifluoroethanol Any one or combination of several.

Further, in step 2, the parts by weight of the components of the first spinning solution are as follows:

Natural source extracellular matrix extract 5-50 parts

Polymer material 50～95 parts

100 parts of solvent.

Further, in step 2, the parts by weight of the components of the second spinning solution are as follows;

Natural source extracellular matrix extract 50-95 parts

Polymer material 5-50 parts

100 parts of solvent.

further,

The composite nanoparticle gel consists of the following components by weight:

Further, the therapeutic factor is any one or a combination of BMP-2, deferoxamine, KGN, VEGF, dexamethasone, eugenol, chondroitin sulfate, and interleukin.

Further, water is ultrapure water.

Further, in the first composite nanofiber yarn, the nanofibers thereof are inclined by 10°-80° along the axial direction of the first composite nanofiber yarn.

Further, in step 2, the preparation method of the first spinning solution and the second spinning solution is:

Step 21, weighing certain parts by weight of natural extracellular matrix extract and polymer material;

Step 22, under the condition of 50°C to 80°C, dissolving the natural source extracellular matrix and the polymer material in the solvent, stirring and dissolving to obtain a clear first spinning solution and a second spinning solution;

Wherein, the mass volume ratio of the first spinning solution and the second spinning solution is 2%-30%.

Further, the method for preparing the first composite nanofiber yarn is:

Connect the first spinning solution to the positive and negative needles respectively, pass the twisted multifilament of polymer material (that is, the yarn reinforcement core) through the beamer, and pre-wrap it on the receiving roller, and then connect the positive and negative needles Connect with a high-voltage power supply for electrospinning to obtain the first nanofiber, and wind the first nanofiber or the second nanofiber on the yarn reinforcing core to obtain the first composite nanofiber yarn (as shown in Fig. 2a-2b).

Further, in step 4, the first composite nanofiber yarn is prepared into a mechanical reinforcement layer by means of doubling, twisting and weaving.

Further, in step 4, the first composite nanofiber yarn is prepared into a mechanical reinforcement layer by means of doubling, twisting and weaving.

Further, in step 4, the first composite nanofiber yarn is prepared into a mechanical reinforcement layer by means of doubling and twisting knitting.

Further, the preparation method of the composite nanoparticle gel is:

Therapeutic factors that promote the proliferation and migration of bone cells and tenocytes are loaded on dendritic inorganic nanoparticles by physical adsorption and chemical grafting, and then the nanoparticles loaded with therapeutic factors are dispersed in dimethyl sulfoxide/ In the poly(ester-ether) polyurethane urea gel with water as the solvent, the composite nanoparticle gel can be obtained (as shown in Figures 3a-3b).

For the prepared multi-level structure bracket, the height of the second bracket 300 is smaller than the height of the first bracket 100 .

Further, the two ends of the second bracket 300 are not in contact with the two ends of the first bracket 100 .

Further, the distance between the first end of the second bracket 300 and the first end of the first bracket 100 is equal to or unequal to the distance between the second end of the second bracket 300 and the second end of the first bracket 100 .

Example 2

This embodiment is a specific embodiment of the present invention.

The preparation method of a kind of multi-level structure support of the present embodiment, comprises the following steps:

Step 1. Washing, pulverizing, degumming, dialysis, and freeze-drying the tussah silkworm chrysalis in sequence, and then spraying 10 to 20 times with phosphate buffer solution containing 0.5% to 1% penicillin/streptomycin double antibody to obtain tussah silkworm pupae Silk fibroin is used as a natural source of extracellular matrix, and it is stored at 4°C to -80°C after freeze-drying;

Step 2, preparation of spinning solution

Step 21, weighing 50 parts by weight of tussah silk fibroin protein and 50 parts by weight of a copolymer of polylactic acid and polycaprolactone;

Step 22. Dissolve the copolymer of tussah silk fibroin, polylactic acid and polycaprolactone in hexafluoroisopropanol under the condition of a water bath at 50°C, and stir to dissolve to obtain a clear first spinning solution and a second clear spinning solution. spinning solution;

Wherein, the mass volume ratio (g/mL) of the first spinning solution and the second spinning solution is 10%;

Step 3, using the first spinning solution to perform trumpet-conjugated electrospinning to prepare the first composite nanofiber yarn;

Step 4, using the four-ply doubling, twisting and weaving technology to use the first composite nanofiber yarn to prepare a mechanical reinforcement layer with a planar structure;

Step 5. Coating composite nanoparticle gel on the front and back of the mechanically enhanced layer to form a gel layer 200, then wrapping to 6.5mm, heat-induced phase separation treatment, freezing at -80°C, and then freeze-drying, Obtain the first support 100 in columnar structure;

Wherein, the composite nanoparticle gel is a poly(ester-ether) polyurethane urethane gel with BMP-2/deferoxamine nanoparticles;

Step 6. Use the second spinning solution to perform trumpet-conjugated electrospinning, and cover the outer surface of the first support 100 with a layer of second composite nanofibers to form the second support 300, so that a polyamide with a diameter of 6 mm can be obtained. Level structure support.

Example 3

This embodiment is a specific embodiment of the present invention.

The preparation method of a kind of multi-level structure support of the present embodiment, comprises the following steps:

Step 1. Washing, pulverizing, degumming, dialysis, and freeze-drying the pigtail in sequence, and then spraying 10 to 20 times with phosphate buffer solution containing 0.5% to 1% penicillin/streptomycin double antibody to obtain pigtail collagen Protein is used as a natural source of extracellular matrix, and it is stored at 4°C to -80°C after freeze-drying;

Step 2, preparation of spinning solution

Step 21, respectively weighing 80 parts by weight of pigtail collagen and 20 parts by weight of poly(p-epoxide dihexanone) as components for preparing the first spinning solution;

Weigh respectively 50 parts by weight of pigtail collagen and 50 parts by weight of poly(p-epoxide dihexanone) as components for preparing the second spinning solution

Step 22. Dissolve pigtail collagen and poly(p-epoxide dihexanone) in tetrahydrofuran/dichloromethane (volume ratio 75:25) in a water bath at 30°C, and stir to dissolve to obtain a clear first spinning solution and a second spinning solution;

Wherein, the mass volume ratio (g/mL) of the first spinning solution and the second spinning solution is 15%;

Step 3, using the first spinning solution to perform trumpet-conjugated electrospinning to prepare the first composite nanofiber yarn;

Step 4, using the four-ply doubling, twisting and weaving technology to use the first composite nanofiber yarn to prepare a mechanical reinforcement layer with a planar structure;

Step 5. Coating composite nanoparticle gel on the front and back of the mechanically enhanced layer to form a gel layer 200, then wrapping to 4.3mm, heat-induced phase separation treatment, freezing at -80°C, and then freeze-drying, Obtain the first support 100 in columnar structure;

Wherein, the composite nanoparticle gel is a poly(ester-ether) polyurethane urethane gel with BMP-2/deferoxamine nanoparticles;

Step 6. Use the second spinning solution to perform trumpet-conjugated electrospinning, and cover the outer surface of the first support 100 with a layer of second composite nanofibers to form the second support 300, so that a polyamide with a diameter of 4 mm can be obtained. Level structure support.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the implementation and protection scope of the present invention. For those skilled in the art, they should be able to realize that all equivalents made by using the description and illustrations of the present invention The solutions obtained by replacement and obvious changes shall all be included in the protection scope of the present invention.

Claims (10)

1. a kind of preparation method of multilevel structure bracket, which comprises the following steps:

Step 1 obtains natural source extracellular matrix, saves at 4 DEG C~-80 DEG C after freeze-drying process；

Step 2 is dissolved the natural source extracellular matrix and high molecular material using solvent, and it is molten to obtain clear first spinning Liquid and the second spinning solution；

Step 3 carries out electrostatic spinning using first spinning solution, to prepare the first composite nano fibre yarn line；

Step 4 prepares the mechanics enhancement layer in planar structure using the first composite nano fibre yarn line；

Step 5, the mechanics enhancement layer obverse and reverse coating composite nanometer particle gel to form gel layer, then carry out Volume, Thermal inactive processing are wrapped up in, the first support for being in the form of a column structure is obtained；

Step 6 carries out loudspeaker-conjugation electrostatic spinning using second spinning solution, covers in the outer surface of the first support One layer of lid the second composite nano fibre yarn line is to form second support, to prepare multilevel structure bracket.

2. the preparation method of multilevel structure bracket according to claim 1, which is characterized in that in step 1, described in acquisition The method of natural source extracellular matrix are as follows:

Pigtail or rat-tail or silkworm chrysalis are successively cleaned, crushed, degumming, dialysis, freeze-drying process, then using containing green The dual anti-phosphate buffer hydro-peening of mycin/streptomysin several times, to obtain the natural source extracellular matrix.

3. the preparation method of multilevel structure bracket according to claim 1, which is characterized in that the high molecular material is Poly- 4 hydroxybutyric acid ester, the copolymer of 3-hydroxybutyrate ester and 3- hydroxyl valerate, polyhydroxyalkanoate, polylactic acid and poly- It is the copolymer of caprolactone, polydioxanone, poly lactide-glycolide acid, any one in biomedical polyurethane Kind or several combinations.

4. the preparation method of multilevel structure bracket according to claim 1, which is characterized in that the solvent is hexafluoro isopropyl Alcohol, N,N-dimethylformamide, DMAC N,N' dimethyl acetamide, tetrahydrofuran, methylene chloride, acetone, chloroform, trifluoroacetic acid, three Any one or the combination of several of them in fluoroethanol.

5. the preparation method of multilevel structure bracket according to claim 1, which is characterized in that described in the step 2 The parts by weight of each component of first spinning solution are as follows:

5~50 parts of natural source extracellular matrix extract

50~95 parts of high molecular material

100 parts of solvent.

6. the preparation method of multilevel structure bracket according to claim 1, which is characterized in that described in the step 2 The parts by weight of each component of second spinning solution are as follows；

50~95 parts of natural source extracellular matrix extract

5~50 parts of high molecular material

100 parts of solvent.

7. the preparation method of multilevel structure bracket according to claim 1, which is characterized in that the composite nanometer particle is solidifying Glue consists of the following parts by weight:

8. the preparation method of multilevel structure bracket according to claim 1, which is characterized in that in first composite Nano In fiber yarn, axially inclined 10 °~80 ° along the first composite nano fibre yarn line of nanofiber.

9. the preparation method of multilevel structure bracket according to claim 1, which is characterized in that described in the step 2 First spinning solution and second spinning solution the preparation method comprises the following steps:

Step 21, the natural source extracellular matrix extract and the high molecular material for weighing constant weight number respectively；

Step 22, under the conditions of 50 DEG C~80 DEG C, the natural source extracellular matrix and the high molecular material are dissolved in described In solvent, stirring and dissolving, to obtain clear first spinning solution and second spinning solution；

Wherein, the mass volume ratio of first spinning solution and second spinning solution is 2%~30%.

10. a kind of multilevel structure bracket is made by the preparation method of any multilevel structure bracket of claim 1~9.