CN1293924C - Tissue engineering peripheral nerve used for repairing peripheral nerve defect and its preparation method - Google Patents

Abstract

The invention belongs to the technical field of medical biomaterials, and relates to a tissue-engineered peripheral nerve product and a preparation method thereof. The tissue-engineered peripheral nerve product is constructed from nerve conduits and glial cells or stem cells. The preparation method includes the preparation of nerve conduits, the preparation of neurotrophic factor-controlled release microspheres, and the construction of tissue-engineered peripheral nerves. The invention has the advantage of having glial cells and neurotrophic factors, which can protect neurons and promote nerve axon regeneration And play an important guiding role in the directionality of axon regeneration.

Description

Tissue engineered peripheral nerve for repairing peripheral nerve defect and preparation method thereof

technical field

The invention belongs to the technical field of medical biomaterials that can be implanted into human bodies, and relates to a tissue-engineered peripheral nerve for repairing peripheral nerve defects and a preparation method thereof.

Background technique

For the long-distance peripheral nerve defects often encountered in clinical treatment, due to the lack of elasticity of the nerves, most cases cannot be directly anastomosed. Autologous nerve transplantation is still the most widely used method in clinical practice. However, this method has disadvantages that are difficult to overcome. After the donor nerve is excised, it will inevitably cause sensory disturbance in the donor area, and traumatic neuroma may occur, causing local pain. Additional nerve extraction will prolong the operation time and increase the scar in the donor area. Moreover, the variation in the thickness of the nerves makes the excised nerves not necessarily suitable for matching, and may cause misaligned regeneration of motor nerves and sensory nerves. In addition, the limited source of donor nerves also greatly limits the application of autologous nerve transplantation.

In the 1980s, Lundborg et al. confirmed the presence of nerve chemotaxis in peripheral nerve regeneration through Y-shaped silicone tube experiments, laying a theoretical foundation for the use of nerve conduits to repair nerve defects. With the development of tissue engineering technology, the use of biomaterials and seed cells to construct tissue-engineered peripheral nerves in vitro to replace autologous nerve transplantation has become a development direction. Applying tissue engineering technology to construct tissue-engineered peripheral nerves, the core of the research is to simulate the natural structure of peripheral nerves, organically combine seed cells and biological scaffold materials to form a structure similar to Büngner ribbons, provide a good growth environment for regenerated nerves, and promote nerve regeneration. regeneration. Tissue engineering repair of peripheral nerve defects can overcome the shortcomings of autologous nerve transplantation and achieve the ideal goal of rapid nerve growth and complete functional recovery.

At present, the tissue-engineered peripheral nerve is still in the stage of animal experiments. The main factor restricting its development is that the number of Schwann cells required as seed cells is very large, but the proliferation of Schwann cells is slow, and it is difficult to culture in vitro, and often contaminates with fibroblasts. .

In addition, studies have shown that neurotrophic factors play an important role in protecting the survival of neurons and promoting nerve regeneration. However, the direct use of neurotrophic factors can only work in the early stages of tissue repair, and it is difficult to maintain the effectiveness of their effects in the later stages; on the one hand, it is difficult to achieve effective concentrations because they are easily diluted and absorbed by body fluids; on the other hand, If the given dose is too large, it will cause the danger of systemic effects, and it will be inactivated due to the environment in the body, so that the desired biological effect cannot be achieved, or it will be exhausted in a short time.

The patent document of the prior art that is relatively close to the present invention is the Chinese patent application of Keihiko Shimizu, Japan published in 2002. This invention mainly relates to the preparation of an artificial neural tube, which is composed of bioabsorbable materials, the surface is covered with collagen, and the inner cavity is It has fine fibrous collagen, and laminin is filled in the gaps. Then there is Wang Shenguo's Chinese patent (application number 00123465.X), the main disadvantages are: (1) The core role played by seed cells and neurotrophic factors in nerve regeneration is ignored, and the promotion of nerve regeneration by materials is overly dependent , the clinical efficacy is uncertain; (2) It does not involve the combination of cells and neurotrophic factors with biomaterials.

Contents of the invention

In view of the current state of the art, the purpose of the present invention is to construct a tissue-engineered peripheral nerve, using glial cells or stem cells that can differentiate into glial cells as seed cells, biodegradable materials as scaffolds, and simultaneously applying a variety of The controlled release system of neurotrophic factors can establish a microenvironment conducive to nerve regeneration, so that nerves can regenerate, and provide a feasible solution for clinical treatment.

The tissue-engineered peripheral nerve used for repairing peripheral nerve defects of the present invention is characterized in that: the tissue-engineered peripheral nerve is constructed by nerve conduits and glial cells or stem cells; the nerve conduits are made of biodegradable materials; The glial cells or stem cells are autologous or allogeneic glial cells or stem cells that can differentiate into nervous system cells; they contain a controlled release system for cells or neurotrophic factors and collagen, laminin (LN), fibronectin (FN) and other extracellular matrix. The biodegradable materials include natural polymer materials such as collagen and chitosan; and polymer synthetic materials such as polylactic acid, polyglycolic acid, and their copolymers; the stem cells include Schwann cells, embryonic stem cells, and adult Stem cells, such as bone marrow mesenchymal stem cells, hematopoietic stem cells, skin stem cells, mesenchymal stem cells, muscle stem cells, liver stem cells, neural stem cells. The nerve guide has micropores of 100 μm-300 μm and a porosity of 50%-90%. The cell concentration contained is 10 ⁵ -10 ⁷ /ml; the neurotrophic factors contained refer to neurotrophic factor NGF, neurotrophic factor-3NT-3, brain-derived neurotrophic factor BDNF, ciliary neurotrophic factor CNTF, One or more of glial cell-derived neurotrophic factor GDNF, transforming growth factor TGFβ family, leukemia inhibitory factor LIF, basic fibroblast growth factor bFGF and insulin-like growth factor IGF. The controlled release system uses biodegradable materials to compound neurotrophic factors, and utilizes the selective permeability of polymers to drugs to achieve controlled release of neurotrophic factors. The diameter of the controlled release microspheres is 100nm-100μm, and the degradation time is About 30 days to 150 days, it can be adjusted according to the speed of nerve regeneration and the distance of regeneration; tissue engineered peripheral nerves are composed of extracellular matrix components such as collagen, laminin (LN) and fibronectin (FN) gel.

The nerve guide of the present invention has a certain degree of permeability, which can diffuse and discharge a large amount of waste produced by metabolism in the process of cell and axon regeneration, prevent a large number of fibroblasts from invading and hinder the passage of nerve growth, and prevent local neurotrophic factors from being released. The loss is conducive to the growth of tiny microvessels in the nearby circulatory system into the catheter.

The neurotrophic factors in the present invention are released by a controlled release system. The main functions of adding trophic factors in the nerve conduit are: inducing stem cells to differentiate into glial cells; maintaining the survival of neurons and promoting the regeneration of nerve axons. However, when nutritional factors are used directly, they are often inactivated due to the environment in the body, and the desired biological effect cannot be achieved, and the release cannot be sustained. We use the controlled release system of cell trophic factors, which can release continuously, maintain the concentration of cell trophic factors, and make them continue to work. The controlled release system uses biodegradable materials to compound neurotrophic factors, so that cell trophic factors are gradually released as the materials degrade. In addition, using the selective permeability of polymers to drugs, neurotrophic factors can be dissolved and diffused in the polymer protective layer at a certain speed, and then enter the body to exert their biological effects, so the biological effects of neurotrophic factors can be improved. Maintain for a long time, so as to realize the controlled release of neurotrophic factors. The controlled-release microspheres of neurotrophic factors used in the present invention have a diameter of 100nm-100μm and a degradation time of 30 days-150 days, which can be adjusted according to the speed and distance of nerve regeneration.

The extracellular matrix filled in the nerve guide of the present invention is the main component of the basement membrane, and can play an important role in guiding the directionality of axon regeneration.

Compared with the prior art, the tissue-engineered peripheral nerve of the present invention has the advantages of adding necessary glial cells and neurotrophic factors in the process of nerve regeneration, and realizing controlled release of neurotrophic factors, which is beneficial to the long-term growth of neurotrophic factors. Through the joint action of glial cells and neurotrophic factors, it can protect neurons and promote nerve axon regeneration; at the same time, tissue engineered peripheral nerves also contain collagen, LN, FN and other extracellular matrices, Plays an important guiding role in the directionality of axon regeneration.

The preparation method of the tissue-engineered peripheral nerve used for repairing the peripheral nerve defect of the present invention includes the preparation method of the nerve guide, the preparation method of the neurotrophic factor controlled release microspheres, the construction method of the tissue-engineered peripheral nerve, and the preparation method of the nerve guide The preparation method is prepared according to the method of controlling solvent volatilization or the method of dissolving salt, and is characterized in that:

1) The steps of the preparation method of the neurotrophic factor controlled release microspheres are as follows:

Dissolve one or several neurotrophic factors and biodegradable materials in a solvent (such as dichloromethane, chloroform, acetic acid, hydrochloric acid, etc.), add dropwise to glycerin, stir to disperse evenly, and pour 0.5% gelatin aqueous solution In, emulsion droplets are dispersed, organic solvents are eluted, and biomaterials are deposited to form microspheres. Collect by centrifugation, wash with distilled water, filter, and dry under normal pressure.

2) The construction method of described tissue engineered peripheral nerve comprises the steps:

Add 1/10 volume of fetal calf serum and 10 times the concentration of minimum essential culture medium DMEM to the solution of extracellular matrix under ice bath, adjust the pH value to 7.2-7.4, seed cells and microspheres compounded with various cell trophic factors , mix well and fill the nerve conduit, after it forms a gel, add DMEM medium for culture.

Description of drawings

Figure 1: Schematic diagram of the tissue-engineered peripheral nerve structure of the present invention.

Among them: 1 nerve stump, 2 nerve conduit, 3 neurotrophic factor NTF. The catheter is filled with a controlled release system of cells and neurotrophic factor NTF and extracellular matrices such as collagen, laminin and fibronectin.

Fig. 2: 12 weeks after tissue engineering nerve repairing of SD rat sciatic nerve defect of 15 mm, the photo of the experimental animal that the regenerated nerve has passed through the defect area.

Detailed ways

Now the present invention will be further described in conjunction with specific embodiments.

1 Schwann cell culture

10 offspring SD rats were sacrificed and disinfected with alcohol. The bilateral sciatic nerves were taken and placed on an ice-bath operating table to remove the epineurium and adhesive tissue. After adding trypsin/collagenase to digest, the upper cell suspension was collected and centrifuged. Clear, add culture medium to resuspend, inoculate in culture flask and culture. After 24 hours, 10 ^-5 M cytarabine was added to the culture medium, and after 48 hours of action, it was replaced with G-418 culture medium containing 100 μg/ml, and cultured continuously for 5 days. Subsequently, 2 μM fluskolin was added to continue culturing, and the medium was changed every 3 days until the cells became confluent.

2 Preparation of the nerve guide

Prepare nerve conduits with pores of 100 μm-300 μm according to controlled solvent evaporation or dissolved salt method, and determine the length of the nerve conduit according to the length of the defect. Generally, the length of the nerve conduit is 5 mm longer than the length of the nerve defect, so that the two ends of the nerve can be socketed.

3 Preparation of Neurotrophic Factor Controlled Release Microspheres

The preparation of the microspheres is carried out in two steps, that is, the dispersion of the organic phase and the elution of the organic solvent. Dissolve one or several neurotrophic factors and biodegradable materials in solvents such as dichloromethane, chloroform, acetic acid, hydrochloric acid, etc., add dropwise to glycerin, stir and disperse evenly, pour into 0.5% gelatin aqueous solution, Emulsion droplets are dispersed, organic solvents are eluted, and biomaterials are deposited to form microspheres. Collect by centrifugation, wash with distilled water, filter, and dry under normal pressure. Using glycerin as a dispersion medium, emulsion droplets with a particle size of less than 30 μm can be obtained at a stirring speed of 500 r/min. The eluent uses 0.5% gelatin aqueous solution, which not only facilitates the diffusion and elution of the solvent, but also prevents the merging of emulsion droplets or the adhesion of microspheres. When the microspheres degrade, the neurotrophic factors are gradually released, and the time can last for 30 days to 150 days.

4 Construction of tissue engineered peripheral nerve

Add 1/10 volume of fetal calf serum and 10 times the concentration of DMEM medium to the solution of extracellular matrix under ice bath, adjust the pH value to 7.2-7.4, mix seed cells and microspheres compounded with various cell trophic factors After uniformity, fill the nerve conduit, and after it forms a gel, add DMEM medium for culture.

5 Repair of sciatic nerve defect in SD rats with tissue engineered peripheral nerve

Tissue-engineered peripheral nerves were used to bridge the sciatic nerve defect, and nerve regeneration and sciatic nerve functional recovery were detected by immunohistochemistry, electrophysiology, transmission electron microscopy, and horseradish peroxidase retrograde tracing. It was found that the nerve fibers were neatly arranged, the number was large, the nerve fibers were thick, the myelin sheath was thick, the interfascicular connective tissue was less, the regenerated myelin sheath was dense, the myelin sheath was obviously thickened, and the axoplasm was rich in neurofilaments and microtubules. Mixed action potentials in the sciatic nerve could be detected, and the sciatic nerve function index showed that the function of the sciatic nerve had largely recovered (see Figure 2).

Claims (4)

1, a kind of tissue engineered peripheral nerve that is used to repair peripheral nerve defection, the stem cell of using the differentiation of neurogliocyte or neurad glial cell is as seed cell, the nerve trachea that adopts Biodegradable material to constitute, use the sustained release microsphere that is compounded with neurotrophic factor simultaneously, and contain extracellular matrix, it is characterized in that: the tissue engineered peripheral nerve conduit has 100 μ m-300 μ m micropores, and porosity is 50%-90%; The cell concentration that is comprised in the tissue engineered peripheral nerve is 10 ⁵-10 ⁷Individual cell/ml; Described sustained release microsphere adopts Biodegradable material compound to neurotrophic factor, and utilize the selection permeability of macromolecule to medicine, realization is to the sustained release of neurotrophic factor, and the sustained release microsphere diameter is 100nm-100 μ m, and degradation time is 30 days-150 days.

2, tissue engineered peripheral nerve according to claim 1 is characterized in that: comprise the gel that is full of collagen, laminin LN and fiber adhesion albumen FN extracellular matrix components formation in the tissue engineered peripheral nerve.

3, tissue engineered peripheral nerve according to claim 1 is characterized in that: described seed cell is a stem cell.

4, according to the preparation method of the described tissue engineered peripheral nerve of claim 1, it is characterized in that: the commercial minimum necessary culture fluid DMEM culture medium that the solution of extracellular matrix is added the hyclone of 1/10 volume, 10 times of concentration under ice bath, regulate pH value to 7.2-7.4, fill with in the nerve trachea with seed cell with after being compounded with the sustained release microsphere mixing of neurotrophic factor, after treating its formation gel, add commercial minimum necessary culture fluid DMEM culture medium culturing; The preparation process of the wherein said sustained release microsphere that is compounded with neurotrophic factor is as follows: one or more neurotrophic factors, Biodegradable material are dissolved in the solvent, be added drop-wise in the glycerol, dispersed with stirring is even, in impouring 0.5% aqueous gelatin solution, disperse emulsion droplet, the eluting organic solvent, biomaterial formation of deposits microsphere, centrifugal collection, distilled water wash filters, and constant pressure and dry is promptly.