CN108939165A - Peripheral nerve scaffold and preparation method thereof - Google Patents

Abstract

The invention discloses a peripheral nerve stent, an outer casing and a filler arranged in the outer casing, the filler is a three-dimensional skeleton formed by curling gauze, and the gauze is made of bio-ink; the invention also discloses the peripheral nerve stent Preparation. The peripheral nerve stent prepared by the method of the present invention has the structure and bionic elements of the natural peripheral nerve; on the basis of ensuring the biocompatibility of the magnesium alloy outer sleeve, the sufficient structural stability of the stent is provided, and the filler can reduce the impact on the stent during the manufacturing process. Ink molding strength requirements expand the range of material selection for fillers, so that the ink can select more biological functions; the present invention uses 3D printed two-dimensional gauze, and then superimposes or curls it as the skeleton of the filler, which can accurately control the structure of the skeleton and its general function. Permeability: The present invention provides fillers with corresponding biological properties, such as promoting axon growth and vascularization, by perfusing or infiltrating specific functional components into the skeleton.

Description

A kind of peripheral nerve support and preparation method thereof

technical field

The invention relates to the technical field of tissue engineering and bionic structures, in particular to a peripheral nerve support and a preparation method thereof.

Background technique

The anatomical structure of the peripheral nerve is complex, which is divided into epineurium area, nerve fascicular area and interfascicular area, etc. There are also functional structures such as microvessels and connective tissue distributed in each area. In addition, the composition and spatial distribution of different regions are also very complex. Nerve repair materials (or catheters) currently reported in the literature are mainly prepared from a single homogeneous material (such as synthetic materials, natural materials, and acellular matrix materials). According to the principle of nerve regeneration, the structure and composition of the catheter have also been bionically designed (such as Gradient distribution of nerve growth factor, etc.), but this kind of scaffold is still relatively simple in composition and structure, and cannot reflect the complex composition and structure of natural nerves.

Compared with traditional processing technologies, bio-3D printing technology has significant advantages in the manufacture of complex tissue structures and the spatial distribution of different components. The most important thing about 3D printing is bio-ink. Although the thermoplastic degradable polyether ester material has a mature printing process and is stable after printing, its mechanical properties are quite different from those of peripheral nerves, and it is difficult to load cells or active proteins under high temperature printing conditions. However, bio-ink materials suitable for cell growth are generally low in strength and poor in stability, and it is difficult to meet the needs of direct printing and stability in later use. At present, there is no relatively complete process for 3D printing peripheral nerve scaffolds using hydrogel ink materials with good biocompatibility.

Contents of the invention

Based on the above problems, the purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide a peripheral nerve scaffold based on magnesium alloy outer structure and 3D printing technology, which has both bioactive function and structural stability.

In order to achieve the above object, the technical solution adopted by the present invention is: a peripheral nerve stent, comprising an outer sleeve and a filler arranged in the outer sleeve, the filler is a three-dimensional skeleton formed by crimping gauze, and the gauze The mesh is made with bio-ink.

Among them, the grid structure of the (two-dimensional) gauze is rectangular, and it can also be adjusted to a prismatic, square, parallelogram, etc. as required. By adjusting the size and shape of the gauze grid, the strength, longitudinal and lateral permeability (provide physical signals to guide the directional growth of axons); the structure of the filler imitates the nerve regeneration area; the three-dimensional skeleton has a porous structure, which is conducive to the immersion of gel materials or solutions with certain biological functions, thereby introducing Specific biological function or microstructure.

Preferably, the outer sleeve is a magnesium alloy sleeve. Among them, the magnesium alloy casing is manufactured by metal processing or magnesium alloy wire braiding, and patents CN103598927 and CN104107096A can be referred to, and its structure imitates the epineurium.

Preferably, the gauze is made by 3D printing.

Preferably, the bio-ink contains at least one of hydrogel prepared by acrylic acid double bond terminal modified PEG, natural material and tissue decellularized hydrogel.

Preferably, said natural material is acrylic or norbornene modified hyaluronic acid, gelatin or collagen.

Preferably, the tissue is peripheral nerves, spinal cord, small intestinal submucosa, umbilical cord, placenta or amniotic membrane. More preferably, the tissue is of human or porcine origin.

Preferably, the three-dimensional framework is perfused or infiltrated with a gel or a solution that can promote axon growth or vascularization. Among them, the gel or solution for promoting vascularization preferably contains VEGF.

Preferably, the three-dimensional framework is perfused or infiltrated with a solution containing β-NGF to promote axon growth; or a hydrogel obtained by decellularizing and pulverizing peripheral nerves from pigs and digesting them with pepsin.

As another aspect of the present invention, the present invention also provides a method for preparing a peripheral nerve scaffold, comprising the steps of:

(1) Provide magnesium alloy outer casing;

(2) Two-dimensional gauze is prepared by using ink with dual sensitivity to temperature and ultraviolet light and 3D printing method, wherein the temperature of the 3D printing barrel is 24-28°C, and the temperature of the forming area is controlled at 4-12°C;

(3) keeping the two-dimensional gauze obtained in step (2) in the above-mentioned low-temperature environment, using 365nm UV radiation with a light intensity of 0.1-3mv/cm2 for 8s-2min; then, by crimping or cutting and superimposing, a three-dimensional skeleton is obtained; Therefore, the polymer in the printed two-dimensional gauze structure can be chemically cross-linked to improve the stability of the printed two-dimensional gauze soft model;

(4) Use a solution of nerve growth factor β-NGF with a concentration of 100pg/ml-10μg/ml or/and a solution of pig-derived peripheral nerves with a mass concentration of 0.1-10% after decellularization, pulverization, and digestion with pepsin. The gel is infiltrated or perfused into the three-dimensional skeleton obtained in step (3), and then filled into the magnesium alloy outer casing to obtain the peripheral nerve scaffold.

Wherein, the ink contains at least one of hydrogel prepared by acrylic acid double bond terminal modified PEG, natural material and tissue decellularized hydrogel; natural material is preferably acrylic acid or norbornene modified hyaluronic acid, gelatin or collagen; in step (2), the two-dimensional gauze model can be designed and printed according to the requirements, and the temperature sensitivity of the ink is initially used for printing; in step (4), the scaffold is biologically modified after infiltration or perfusion to Conducive to the extension and growth of nerve axons.

In summary, the beneficial effects of the present invention are:

(1) the peripheral nerve support of the present invention has the structure of the natural peripheral nerve and the bionic elements of composition;

(2) On the basis of ensuring biocompatibility, the magnesium alloy outer sleeve provides sufficient structural stability of the stent. During the manufacturing process, the filler can reduce the requirements for the forming strength of the ink, and expand the material selection range of the filler, so that the ink can choose more. biological function;

(3) Using 3D printed two-dimensional gauze, and then superimposing or curling it as the filler skeleton, can accurately control the structure and permeability of the skeleton;

(4) The present invention provides fillers with corresponding biological characteristics, such as promoting axon growth and vascularization, by perfusing or infiltrating specific functional components into the skeleton;

(5) The structure and preparation method of the present invention are simple and easy to realize.

Description of drawings

Fig. 1 is the preparation flow schematic diagram of peripheral nerve support of the present invention;

Wherein, 1. outer sleeve, 2. three-dimensional skeleton, 3. gauze, 4. 3D printer.

Detailed ways

The inventor of the present application uses 3D printing technology to manufacture a two-dimensional gauze structure, in which the structural units can be designed into different shapes according to requirements, and then the preparation of the three-dimensional skeleton is realized by curling or stacking; in order to maintain the internal environment of the printed three-dimensional skeleton structure stability, reduce the generation of scars, and improve the operability of clinical applications. The 3D printed skeleton structure and the magnesium alloy outer sleeve are assembled into a scaffold with a tube core structure; in order to improve the biological function of the scaffold, it can be further perfused or infiltrated with A gel material or solution with certain biological functions.

The 3D printing filler of this application is a soft gel ink material that can promote the growth of axons. The self-support and stability of the 0-5cm slender peripheral nerve scaffolds printed with this ink cannot meet the test and clinical requirements. The requirements of structural stability, therefore, match with the magnesium alloy outer casing, place the 3D printing filler in the outer casing, and assemble it into a peripheral nerve scaffold with a tube core structure.

This application uses 3D printing technology to prepare a peripheral nerve scaffold with both biological functions and structural stability through bionic technology; specifically, the peripheral nerve scaffold of the present invention has the following advantages:

1. The structure is bionic, the outer sleeve is to maintain the shape stability of the stent and facilitate the clinical suturing operation, and the filler provides a microenvironment for nerve regeneration;

2. The outer casing is made of magnesium alloy, which takes into account both biocompatibility and structural stability; magnesium alloy is easy to process and can precisely adjust parameters, such as magnesium alloy composition, porous structure pore diameter, density, etc.;

3. Select soft materials with better biocompatibility that match the mechanical properties of peripheral nerves as 3D printing inks. Peripheral nerve catheters prepared by traditional techniques must meet certain mechanical properties to maintain stability. Therefore, their mechanical properties are generally far superior higher than the natural peripheral nerves. In addition, the composite ink in the present invention includes a modified gel and peripheral nerve decellularized tissue hydrogel that are conducive to shaping, taking into account 3D printable properties and specific biological activities;

4. Since the tissue engineering scaffold should fully consider the transportation of nutrients, the design of the structural unit in this application can adjust the permeability of the material (through the grid), which is conducive to the transportation of substances; in addition, the three-dimensional skeleton can load biological signals uniformly or gradiently Molecules, realize the spatial distribution of signals, and guide the directional extension of axons;

5. The structure and permeability of the bracket can be adjusted according to the needs;

6. The three-dimensional framework of the present invention can also add bioactive molecules that can promote axonal growth activity or increase the speed of blood vessel growth, so as to improve the survival and proliferation of early cells and facilitate the repair of long-segment nerve defects.

In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified, the technologies in this application, such as the preparation of magnesium alloy sleeves and 3D printing technology, all belong to the well-known technologies in this field. Creative work can realize the corresponding structure or product.

Example 1

As shown in Figure 1, an embodiment of the peripheral nerve stent of the present invention comprises an outer casing 1 and a filler disposed in the outer casing 1, the filler is a three-dimensional skeleton 2 formed by crimping a gauze 3, and the gauze 3 Made of bio-ink, the outer sleeve 1 is a magnesium alloy sleeve, and the gauze 3 is made by 3D printing.

Wherein, the bio-ink is a hydrogel prepared by modifying PEG with double bond end groups of acrylic acid; the three-dimensional skeleton is perfused or infiltrated with a solution that can promote axon growth, which contains β-NGF.

The preparation method of above-mentioned peripheral nerve stent, comprises the steps:

(1) Provide magnesium alloy outer casing;

(2) Two-dimensional gauze was prepared by using an ink with dual sensitivity to temperature and ultraviolet light (bio-ink) and a 3D printing method, wherein the temperature of the 3D printing barrel was 24°C, and the temperature of the forming area was controlled at 6°C;

(3) keeping the two-dimensional gauze obtained in step ( ² ) in the above-mentioned low-temperature environment, using 365nm UV radiation with a light intensity of 3mv/cm for 8s; then, by curling or cutting and superimposing, a three-dimensional skeleton is obtained;

(4) Infiltrate or perfuse the three-dimensional framework obtained in step (3) with a solution of nerve growth factor β-NGF with a concentration of 100 pg/ml, and then fill it into the magnesium alloy outer sleeve to prepare the peripheral nerve scaffold.

Afterwards, the peripheral nerve scaffold was freeze-dried, and the macroscopic and microstructure of the peripheral nerve scaffold was observed by scanning electron microscopy (SEM); the peripheral nerve scaffold was placed in PBS at 37°C to continuously observe its shape stability, and the results showed that the Peripheral nerve scaffolds can exist stably in PBS for more than 6 days.

Example 2

An embodiment of the peripheral nerve stent of the present invention includes an outer tube 1 and a filler disposed in the outer tube 1, the filler is a three-dimensional skeleton 2 formed by crimping a gauze 3, the gauze 3 is made of bio-ink, The outer sleeve 1 is a magnesium alloy sleeve, and the gauze is made by 3D printing.

Among them, the bio-ink is hyaluronic acid modified by acrylic acid or norbornene; the three-dimensional framework is perfused or infiltrated with a hydrogel obtained by decellularizing and pulverizing peripheral nerves from pigs and digesting them with pepsin.

The preparation method of above-mentioned peripheral nerve stent, comprises the steps:

(1) Provide magnesium alloy outer casing;

(2) Two-dimensional gauze was prepared by using ink with dual sensitivity to temperature and ultraviolet light (bio-ink) and 3D printing method, wherein the temperature of the 3D printing barrel was 25°C, and the temperature of the forming area was controlled at 4°C;

(3) keeping the two-dimensional gauze obtained in step ( ² ) in the above-mentioned low-temperature environment, using 365nmUV irradiation with a light intensity of 1.5mv/cm for 25s; then, by crimping or cutting and superimposing, a three-dimensional skeleton is obtained;

(4) Infiltrate or perfuse the hydrogel obtained by pepsin digestion with the pig-derived peripheral nerve with a mass concentration of 0.1-10% after decellularization and crushing into the three-dimensional skeleton obtained in step (3), and then fill it into the magnesium alloy In the outer cannula, the peripheral nerve scaffold is prepared.

Afterwards, the peripheral nerve scaffold was freeze-dried, and the macroscopic and microstructure of the peripheral nerve scaffold was observed by scanning electron microscopy (SEM); the peripheral nerve scaffold was placed in PBS at 37°C to continuously observe its shape stability, and the results showed that the Peripheral nerve scaffolds can exist stably in PBS for more than 7 days.

Example 3

An embodiment of the peripheral nerve stent of the present invention includes an outer sleeve 1 and a filler disposed in the outer sleeve 1, the filler is a three-dimensional skeleton 2 formed by crimping gauze, the gauze 3 is made of bio-ink, the outer sleeve Tube 1 is a magnesium alloy casing, and the gauze is made by 3D printing.

Among them, the bio-ink is tissue decellularized hydrogel; the tissue is pig-derived peripheral nerve, spinal cord, small intestinal submucosa, umbilical cord, placenta or amniotic membrane; the three-dimensional skeleton is perfused or infiltrated with β-NGF containing β-NGF that can promote axon growth. solution.

The preparation method of above-mentioned peripheral nerve stent, comprises the steps:

(1) Provide magnesium alloy outer casing;

(2) Two-dimensional gauze was prepared by using an ink with dual sensitivity to temperature and ultraviolet light (bio-ink) and a 3D printing method, wherein the temperature of the 3D printing barrel was 27°C, and the temperature of the forming area was controlled at 12°C;

(3) keeping the two-dimensional gauze obtained in step ( ² ) in the above-mentioned low-temperature environment, and using 365nmUV irradiation with a light intensity of 0.7mv/cm for 1min; then, by crimping or cutting and superimposing, a three-dimensional skeleton is obtained;

(4) Infiltrate or perfuse the three-dimensional framework obtained in step (3) with a solution of nerve growth factor β-NGF with a concentration of 1 μg/ml, and then fill it into the magnesium alloy outer sleeve to prepare the peripheral nerve scaffold.

Afterwards, the peripheral nerve scaffold was freeze-dried, and the macroscopic and microstructure of the peripheral nerve scaffold was observed by scanning electron microscopy (SEM); the peripheral nerve scaffold was placed in PBS at 37°C to continuously observe its shape stability, and the results showed that the Peripheral nerve scaffolds can exist stably in PBS for more than 12 days.

Example 4

An embodiment of the peripheral nerve stent of the present invention includes an outer tube 1 and a filler disposed in the outer tube 1, the filler is a three-dimensional skeleton 2 formed by crimping a gauze 3, the gauze 3 is made of bio-ink, The outer sleeve 1 is a magnesium alloy sleeve, and the gauze 3 is made by 3D printing.

Among them, the bio-ink contains hydrogels prepared by acrylic acid double bond terminal modified PEG and tissue decellularized hydrogels; the tissues are pig-derived peripheral nerves, spinal cord, small intestinal submucosa, umbilical cord, placenta or amniotic membrane; perfusion on the three-dimensional skeleton Or infiltrated with a solution containing β-NGF that can promote axon growth, and a hydrogel obtained by decellularizing and pulverizing peripheral nerves from pigs and digesting them with pepsin.

The preparation method of above-mentioned peripheral nerve stent, comprises the steps:

(1) Provide magnesium alloy outer casing;

(2) Two-dimensional gauze was prepared by using ink with dual sensitivity to temperature and ultraviolet light (bio-ink) and 3D printing method, wherein the temperature of the 3D printing barrel was 28°C, and the temperature of the forming area was controlled at 9°C;

(3) keeping the two-dimensional gauze obtained in step (2) in the above-mentioned low-temperature environment, using 365nmUV irradiation with a light intensity of 0.1mv/cm for ^2min ; then, by crimping or cutting and superimposing, a three-dimensional skeleton is obtained;

(4) Use a solution of nerve growth factor β-NGF with a concentration of 10 μg/ml and a pig-derived peripheral nerve with a mass concentration of 0.1 to 10% to infiltrate or infuse the hydrogel obtained by decellularization, pulverization, and digestion with pepsin. The three-dimensional framework obtained in step (3) is then filled into the magnesium alloy outer casing to obtain the peripheral nerve support.

Afterwards, the peripheral nerve scaffold was freeze-dried, and the macroscopic and microstructure of the peripheral nerve scaffold was observed by scanning electron microscopy (SEM); the peripheral nerve scaffold was placed in PBS at 37°C to continuously observe its shape stability, and the results showed that the Peripheral nerve scaffolds can exist stably in PBS for more than 8 days.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that The technical solution of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention.

Claims (9)

1. a kind of peripheral nerve bracket, which is characterized in that the peripheral nerve bracket include outer tube and be set to the outer tube Interior filler, the filler are three-dimensional frameworks made of gauze curling, and the gauze is made of bio-ink.

2. peripheral nerve bracket according to claim 1, which is characterized in that the outer tube is magnesium alloy casing.

3. peripheral nerve bracket according to claim 1, which is characterized in that the gauze is made of 3D printing method.

4. peripheral nerve bracket according to claim 1, which is characterized in that the bio-ink contains acrylic double bond end At least one of hydrogel, natural material and the de- cell hydrogel of tissue of base modified PE G preparation.

5. peripheral nerve bracket according to claim 4, which is characterized in that the natural material is acrylic acid or norborneol Alkene modified hyaluronic acid, gelatin or collagen.

6. peripheral nerve bracket according to claim 4, which is characterized in that the tissue is peripheral nerve, spinal cord, small intestine Submucosa, umbilical cord, placenta or amnion.

7. peripheral nerve bracket according to claim 1, which is characterized in that being perfused or be impregnated on the three-dimensional framework can Promote the gel or solution of axon growth or vascularization.

8. peripheral nerve bracket according to claim 1 or claim 7, which is characterized in that be perfused or infiltrate on the three-dimensional framework The water for thering is the peripheral nerve in solution or the pig source containing β-NGF to obtain after taking off cell, crushing through pepsin digestion Gel.

9. a kind of preparation method of peripheral nerve bracket, which comprises the steps of:

(1) magnesium alloy outer tube is provided；

(2) two-dimentional gauze is prepared using with temperature, the ink of ultraviolet light sensitive characteristic and 3D printing method, wherein 3D is beaten Printing barrel temperature is 24~28 DEG C, and the control of forming area temperature is 4~12 DEG C；

(3) two-dimentional gauze obtained by step (2) is kept to use light intensity for 0.1~3mv/cm in above-mentioned low temperature environment²365nm UV Irradiate 8s~2min；Then, by crimping or cutting superposition, three-dimensional framework is made；

(4) using concentration for the solution of the nervegrowthfactor-β-NGF of the μ of 100pg/ml~10 g/ml or/and mass concentration is 0.1 The hydrogel infiltration or perfusion that~10% pig source peripheral nerve obtains after taking off cell, crushing through pepsin digestion To three-dimensional framework obtained by step (3), then it is loaded into the magnesium alloy outer tube, the peripheral nerve bracket is made.