Preparation method of biological annulus fibrosus

Abstract

The present invention relates to a method for preparing a biological annulus fibrosus. This method can achieve integrated processing of materials and cells and directly prepare a biological annulus fibrosus by using the electrospinning process. The specific steps are as follows: (1) Prepare the materials sprayed on the rotating device with a complete culture medium and prepare a water-soluble degradable polymer into a uniform solution with deionized water; (2) Divide the degradable polymer solution into three equal parts after high-temperature steam sterilization; (3) Add the outer cell suspension and the inner cell suspension into two of the above-mentioned sterilized materials respectively and stir evenly; (4) Use three nozzles to extract the above three materials respectively and place them on a micropump according to the preparation sequence, turn on the high-voltage power supply and the micropump, set the corresponding parameters, and perform electrospraying to finally obtain a biological annulus fibrosus. The method of the present technical invention is simple and easy to implement, and the biological annulus fibrosus has great practical significance for the treatment of lumbar disc herniation.

Description

Technical Field

[0001] The present invention relates to a method for preparing a biological annulus fibrosus, and belongs to the fields of preparation of tissue engineering annulus fibrosus and treatment of intervertebral disc herniation. Background Art

[0002] Intervertebral disc herniation easily leads to loss of labor, and treatment measures such as resection of the intervertebral disc and bone graft fusion fixation can only relieve symptoms temporarily, and recurrence also occurs after surgery and cannot be cured. The emergence of tissue engineering technology provides new ideas and methods for the treatment of intervertebral disc herniation, that is, to manufacture a biological annulus fibrosus to replace those broken natural annulus fibrosus and prevent the nucleus pulposus from prolapsing.

[0003] How to make the manufactured biological annulus fibrosus continuously contain proteoglycans and collagen fibers to provide the necessary substances for ensuring the normal physiological metabolism of the natural regenerated annulus fibrosus in the future is the key problem to be solved in the manufacture of the biological annulus fibrosus.

[0004] Electrospinning technology can form a nanofiber scaffold containing cells. Based on the advantages of this technology, the present invention uses this technology to prepare a biological annulus fibrosus with a concentric circular arrangement of outer, middle and inner layers that mimics the natural annulus fibrosus, and different cells are contained in the inner and outer layers of the annulus. Cells grow on the biological annulus fibrosus and synthesize and secrete proteoglycans and collagen fibers, gradually making the outer layer mainly composed of collagen fibers and the inner layer forming a fibrocartilage zone. Summary of the Invention

[0005] The purpose of the present invention is to solve the above problems, provide a method for preparing a biological annulus fibrosus, utilize electrospinning technology, and focus on constructing a biological annulus fibrosus with a concentric circular arrangement of outer, middle and inner layers containing cells to provide more advanced technical support for the treatment of intervertebral disc herniation.

[0006] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A method for preparing a biological annulus fibrosus, the annulus fibrosus is composed of a biodegradable polymer and cells, and is prepared by an electrospinning process, including the following steps:

[0008] 1) Weigh 2.0 - 2.5 grams of Pluronic F-127 material, add it to 8.0 - 10.0 grams of pre-cooled complete medium, place it on a shaker at 4°C for 24 hours until fully dissolved, then filter the Pluronic F-127 solution with a 0.22 mm filter, place it in a 4°C refrigerator for 2 hours, and then spray it on a rotating device. After solidification at room temperature, it is reserved for use;

[0009] 2) Weigh 1.5 - 2.0 grams of polyvinyl alcohol (PVA) material, and slowly add it to 15 - 20 grams of deionized water under stirring. After sufficient swelling, heat it up to about 80 - 100 °C to accelerate dissolution, and keep it warm for 3 hours to obtain a uniform polymer solution. Then, sterilize it with high-temperature steam at 100 °C, divide it into three equal parts, and put one part into nozzle two for standby;

[0010] 3) Digest the cells of the 3rd generation New Zealand white rabbits with 0.25% trypsin, collect them in a 50 mL centrifuge tube, centrifuge at 1000 r / min for 5 min, discard the supernatant, transfer the cell suspension to a sterilized portion of the PVA material, stir evenly to obtain a PVA solution containing cells, and put it into nozzle one for standby;

[0011] 4) Digest the cells of the 4th generation New Zealand white rabbits with 0.25% trypsin, collect them in a 50 mL centrifuge tube, centrifuge at 1000 r / min for 5 min, discard the supernatant, transfer the cell suspension to a sterilized portion of the PVA material, stir evenly to obtain a PVA solution containing cells, and put it into nozzle three for standby;

[0012] 5) Apply a DC voltage of 6 - 8 kV to nozzle one, the distance between the nozzle and the rotating device is 6 - 8 mm, and the negative electrode is loaded on the receiving platform. Extrude the material continuously at a stable flow rate of 150 - 180 μL / min provided by a micro pump, and the rotating device rotates at a speed of 800 r / min for 1 - 3 hours. The nanofibers wrap the solidified Pluronic F - 127 material to complete the preparation of the inner layer of the biofiber ring;

[0013] 6) Apply a DC voltage of 10 - 13 kV to nozzle two, the distance between the nozzle and the rotating device is 15 - 20 mm, and the negative electrode is loaded on the receiving platform. Extrude the material continuously at a stable flow rate of 350 - 380 μL / min provided by a micro pump, and the rotating device rotates at a speed of 1000 r / min for 1 - 2 hours to complete the preparation of the middle layer of the biofiber ring;

[0014] 7) Apply a DC voltage of 6 - 8 kV to nozzle three, the distance between the nozzle and the rotating device is 6 - 8 mm, and the negative electrode is loaded on the receiving platform. Extrude the material continuously at a stable flow rate of 150 - 180 μL / min provided by a micro pump, and the rotating device rotates at a speed of 800 r / min for 1 - 3 hours to complete the preparation of the outer layer of the biofiber ring;

[0015] 8) Place the rotating device in a 4 °C refrigerator for 1 - 2 hours, the Pluronic F - 127 material melts, and take out the biofiber ring that has fallen off the rotating device.

[0016] The cells in step 3) are chondrocytes or osteocytes.

[0017] The cells in step 4) are muscle cells or adipose stem cells.

[0018] Compared with the prior art, the present invention has the following obvious and prominent substantive features and remarkable advantages:

[0019] The biological annulus fibrosus of the present invention has a concentric circle shape and has a three-layer structure; the fiber thicknesses of the middle layer and the inner and outer layers are different, and the outer layer and the inner layer are respectively prepared from different cells and biodegradable materials; the overall structure of the biological annulus fibrosus simulates the natural annulus fibrosus, shortening the treatment time of intervertebral disc herniation. This method has the advantages of simple process, good controllability and high efficiency. Description of the Drawings

[0020] Figure 1 It is a schematic diagram of the biological annulus fibrosus preparation system.

[0021] Figure 2 It is a schematic diagram of the electrospinning process of the biological annulus fibrosus.

[0022] Figure 3 It is a schematic diagram of the biological annulus fibrosus structure. Detailed Description of the Invention

[0023] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings:

[0024] As Figure 1 shown is a schematic diagram of the biological annulus fibrosus preparation system. Among them: The computer control system 1 is connected to the controller 2, the controller 2 is connected to the high-voltage power supply 6 and the micro pump 3, the micro pump 3 is connected to the nozzle 4, the positive pole of the high-voltage power supply 6 is loaded on the needle of the nozzle 4, the negative pole is loaded on the receiving platform 5, and the rotating device 7 is installed on the platform 5.

[0025] Example 1

[0026] A method for preparing a biological annulus fibrosus, the process for preparing the biological annulus fibrosus includes the following steps:

[0027] 1) Weigh 2.5 grams of Pluronic F-127 material, add it to 10.0 grams of pre-cooled complete culture medium, place it on a shaker at 4 °C for 24 hours until fully dissolved, then filter the Pluronic F-127 solution with a 0.22 mm filter, place it in a 4 °C refrigerator for 2 hours, and then spray it on the rotating device, and wait for it to solidify at room temperature for later use;

[0028] 2) Weigh 2.0 grams of polyvinyl alcohol (PVA) material, and slowly add it to 20.0 grams of deionized water under stirring. After sufficient swelling, heat it up to about 100 °C to accelerate dissolution, and keep it warm for 3 hours to obtain a uniform polymer solution. Then, sterilize it with high-temperature steam at 100 °C, divide it into three equal parts, and put one part into nozzle two for standby;

[0029] 3) Digest the chondrocytes of the 3rd generation of New Zealand white rabbits with 0.25% trypsin, collect them in a 50 mL centrifuge tube, centrifuge at 1000 r / min for 5 min, discard the supernatant, transfer the cell suspension to a sterilized portion of PVA material, stir evenly to obtain a PVA solution containing cells, and put it into nozzle one for standby;

[0030] 4) Digest the myocytes of the 4th generation of New Zealand white rabbits with 0.25% trypsin, collect them in a 50 mL centrifuge tube, centrifuge at 1000 r / min for 5 min, discard the supernatant, transfer the cell suspension to a sterilized portion of PVA material, stir evenly to obtain a PVA solution containing cells, and put it into nozzle three for standby;

[0031] 5) Apply a DC voltage of 6 kV to nozzle one, the distance between the nozzle and the rotating device is 6 mm, and the negative electrode is applied on the receiving platform. Extrude the material continuously at a stable flow rate of 150 μL / min provided by a micro-pump, and the rotating device rotates at a speed of 800 r / min for 1 hour of spinning. The nanofibers wrap the solidified Pluronic F-127 material to complete the preparation of the inner layer of the biofiber ring;

[0032] 6) Apply a DC voltage of 13 kV to nozzle two, the distance between the nozzle and the rotating device is 20 mm, and the negative electrode is applied on the receiving platform. Extrude the material continuously at a stable flow rate of 380 μL / min provided by a micro-pump, and the rotating device rotates at a speed of 1000 r / min for 1 hour of spinning to complete the preparation of the middle layer of the biofiber ring;

[0033] 7) Apply a DC voltage of 6 kV to nozzle three, the distance between the nozzle and the rotating device is 6 mm, and the negative electrode is applied on the receiving platform. Extrude the material continuously at a stable flow rate of 150 μL / min provided by a micro-pump, and the rotating device rotates at a speed of 800 r / min for 1 hour of spinning to complete the preparation of the outer layer of the biofiber ring;

[0034] 8) Place the rotating device in a 4 °C refrigerator for 1 hour, the Pluronic F-127 material melts, and take out the biofiber ring that has fallen off the rotating device.

[0035] Example 2

[0036] This example is basically the same as Example 1, except that: the cells used in step 3) are osteocytes, and the cells used in step 4) are adipose stem cells.

Claims

1. Method for preparing biological annulus fibrosus, the annulus fibrosus being composed of a biodegradable polymer and cells, prepared by electrospinning process, comprising the following steps: 1) Weigh 2.5 grams of Pluronic F-127 material, add it to 10.0 grams of pre-cooled complete medium, place it in a shaker at 4°C for 24 hours until fully dissolved, then filter the Pluronic F-127 solution with a 0.22 mm filter, place it in a 4°C refrigerator for 2 hours, and then spray it on a rotating device. After curing at room temperature, it is ready for use; 2) Weigh 2.0 grams of polyvinyl alcohol (PVA) material, slowly add it to 20.0 grams of deionized water under stirring until fully swollen, then heat it to 100°C to accelerate dissolution and keep it warm for 3 hours to obtain a uniform polymer solution. Then, sterilize it with high-temperature steam at 100°C and divide it into three equal parts. Load one part into nozzle two for standby; 3) Digest the cells of the 3rd generation New Zealand white rabbits with 0.25% trypsin, collect them in a 50 mL centrifuge tube, centrifuge at 1000 r / min for 5 min, discard the supernatant, transfer the cell suspension to a sterilized portion of PVA material, stir evenly to obtain a PVA solution containing cells, and load it into nozzle one for standby; 4) Digest the cells of the 4th generation New Zealand white rabbits with 0.25% trypsin, collect them in a 50 mL centrifuge tube, centrifuge at 1000 r / min for 5 min, discard the supernatant, transfer the cell suspension to a sterilized portion of PVA material, stir evenly to obtain a PVA solution containing cells, and load it into nozzle three for standby; 5) Apply a DC voltage of 6 kV to nozzle one, the distance between the nozzle and the rotating device is 6 mm, the negative electrode is loaded on the receiving platform, continuously extrude the material at a stable flow rate of 150 μL / min provided by a micro pump, the rotating device rotates at a speed of 800 r / min, and electrospin for 1 hour. The nanofibers wrap the solidified Pluronic F-127 material to complete the preparation of the inner layer of the biological annulus fibrosus; 6) Apply a DC voltage of 13 kV to nozzle two, the distance between the nozzle and the rotating device is 20 mm, the negative electrode is loaded on the receiving platform, continuously extrude the material at a stable flow rate of 380 μL / min provided by a micro pump, the rotating device rotates at a speed of 1000 r / min, and electrospin for 1 hour to complete the preparation of the middle layer of the biological annulus fibrosus; 7) Apply a DC voltage of 6 kV to nozzle three, the distance between the nozzle and the rotating device is 6 mm, the negative electrode is loaded on the receiving platform, continuously extrude the material at a stable flow rate of 150 μL / min provided by a micro pump, the rotating device rotates at a speed of 800 r / min, and electrospin for 1 hour to complete the preparation of the outer layer of the biological annulus fibrosus; 8) Place the rotating device in a 4°C refrigerator for 1 hour, the Pluronic F-127 material melts, and take out the biological annulus fibrosus that has fallen off the rotating device.

2. The preparation method of the biological annulus fibrosus according to claim 1, characterized in that: The cells in step 3) are chondrocytes or osteocytes, and the cells used in step 4) are myocytes or adipose stem cells.

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