阅读说明：本技术 生物纤维环制备方法 (Preparation method of biological fiber ring ) 是由 陈海萍 谢世坤 纪军 吴素蓉 潘维东 邹杰慧 于 2020-06-09 设计创作，主要内容包括：本发明涉及生物纤维环制备方法。该方法可实现材料和细胞集成加工,直接利用静电纺丝工艺制备生物纤维环。具体步骤为：(1)用完全培养基配制喷洒在旋转装置上的材料、用去离子水配制水溶性可降解高分子聚合物成均一溶液；(2)将可降解高分子聚合物溶液高温蒸汽消毒后均分三份；(3)将外层细胞悬液、内层细胞悬液分别加入到上述消毒后的二份材料里面,搅拌均匀；(4)用三个喷头分别抽取上述三份材料按制备顺序置于微量泵上,开启高压电源和微量泵,设置好相应的参数,进行电喷,最后得到生物纤维环。本技术发明方法简单易行,生物纤维环对椎间盘突出治疗具有重大的现实意义。(The invention relates to a preparation method of a biological fiber ring. The method can realize integrated processing of materials and cells, and directly utilizes the electrostatic spinning process to prepare the biological fiber ring. The method comprises the following specific steps: (1) preparing a material sprayed on a rotating device by using a complete culture medium, and preparing a water-soluble degradable high-molecular polymer into a uniform solution by using deionized water; (2) sterilizing degradable high molecular polymer solution with high-temperature steam, and dividing into three parts; (3) respectively adding the outer-layer cell suspension and the inner-layer cell suspension into the two sterilized materials, and uniformly stirring; (4) and (3) respectively pumping the three materials by using three nozzles, placing the three materials on a micro pump according to the preparation sequence, starting a high-voltage power supply and the micro pump, setting corresponding parameters, and performing electric spraying to obtain the biological fiber ring. The method of the invention is simple and easy to implement, and the biological fiber ring has great practical significance for treating the herniated disk.)

1. The preparation method of the biological fiber ring, the fiber ring is composed of degradable high molecular polymer and cells, is prepared by utilizing an electrostatic spinning process, and comprises the following steps:

1) weighing 2.5 g of Pluronic F-127(Pluronic F-127) material, adding into 10.0 g of precooled complete culture medium, placing in a shaking table at 4 ℃ for 24 hours until the Pluronic F-127 material is fully dissolved, filtering the Pluronic F-127 solution by using a 0.22mm filter, placing in a refrigerator at 4 ℃ for 2 hours, spraying on a rotating device, and curing at room temperature for later use;

2) weighing 2.0 g of polyvinyl alcohol (PVA) material, slowly adding the PVA material into 20.0 g of deionized water under the condition of stirring, heating to about 100 ℃ after full swelling, accelerating dissolution, preserving heat for 3 hours to prepare uniform high molecular solution, sterilizing by high-temperature steam at 100 ℃, uniformly dividing into three parts, and filling one part into a second spray head for later use;

3) digesting cells of 3 rd generation New Zealand white rabbits with 0.25% trypsin, collecting the cells in a 50mL centrifuge tube, centrifuging for 5min at 1000r/min, discarding the supernatant, transferring the cell suspension to a sterilized PVA material, uniformly stirring to obtain a PVA solution containing cells, and filling the PVA solution into a first spray nozzle for later use;

4) digesting cells of a 4 th generation New Zealand white rabbit by using 0.25% trypsin, collecting the cells in a 50mL centrifuge tube, centrifuging for 5min at 1000r/min, removing supernatant, transferring cell suspension to a disinfected PVA material, uniformly stirring to obtain a PVA solution containing cells, and filling the PVA solution into a third nozzle for later use;

5) and 6kV direct-current voltage is loaded on the first spray head, the distance between the spray head and the rotating device is 6mm, and the negative electrode is loaded on the receiving platform. Continuously extruding the material at a constant flow rate of 150 mu L/min provided by a micro pump, rotating a rotating device at a speed of 800r/min, spinning for 1 hour, and wrapping the cured Pluronic F-127 material by the nano fiber to finish the preparation of the inner layer of the biological fiber ring;

6) and a 13kV direct-current voltage is loaded on the second spray head, the distance between the spray head and the rotating device is 20mm, and the negative electrode is loaded on the receiving platform. Continuously extruding the material at a stable flow rate of 380 μ L/min provided by a micro pump, rotating a rotating device at a speed of 1000r/min, and spinning for 1 hour to complete the preparation of the middle layer of the biological fiber ring;

7) and 6kV direct-current voltage is loaded on the third spray head, the distance between the spray head and the rotating device is 6mm, and the negative electrode is loaded on the receiving platform. Continuously extruding the material at a stable flow rate of 150 mu L/min provided by a micro pump, rotating a rotating device at a speed of 800r/min, and spinning for 1 hour to finish the preparation of the outer layer of the biological fiber ring;

8) the rotary device was placed in a refrigerator at 4 ℃ for 1 hour, the Pluronic F-127 material melted, and the bio-fiber rings that had fallen off the rotary device were removed.

2. The method for preparing a biofabric ring according to claim 1, wherein: the cells in the step 3) are chondrocytes or osteocytes, and the cells adopted in the step 4) are myocytes or adipose-derived stem cells.

Technical Field

The invention relates to a preparation method of a biological fiber ring, belonging to the field of tissue engineering fiber ring preparation and treatment of intervertebral disc herniation.

Background

The herniation of intervertebral disc easily causes the loss of labor force, and the treatment measures such as the removal of intervertebral disc, the fusion and fixation of bone grafting and the like can only temporarily relieve the symptoms, and the postoperative recurrence can not be cured radically. The advent of tissue engineering technology has provided new ideas and methods for the treatment of disc herniation by creating a biological annulus to replace those natural annuli that have been ruptured to prevent herniation of the nucleus pulposus.

How to make the produced biological fiber ring continuously contain proteoglycan and collagen fiber, which provides the necessary substances for ensuring the normal physiological metabolic activity of the natural regenerated fiber ring, is the key problem to be solved for producing the biological fiber ring.

The electrostatic spinning technology can form the nanofiber scaffold containing cells, and based on the advantages of the technology, the invention adopts the technology to prepare the biological fiber ring which simulates the concentric circular arrangement of the outer layer, the middle layer and the inner layer of a natural fiber ring, wherein the inner layer and the outer layer of the ring contain different cells. Cells grow on the biological fiber ring to synthesize and secrete proteoglycan and collagen fiber, so that the outer layer is mainly composed of collagen fiber components, and the inner layer forms a fibrocartilage band.

Disclosure of Invention

The invention aims to solve the problems and provides a preparation method of a biological fiber ring, which aims to construct an outer layer, a middle layer and an inner layer of the biological fiber ring which contain cells and are arranged in concentric circles by utilizing an electrostatic spinning technology and provide more advanced technical support for treating the protrusion of intervertebral disc.

In order to achieve the purpose, the invention adopts the following technical scheme:

the preparation method of the biological fiber ring, the fiber ring is composed of degradable high molecular polymer and cells, is prepared by utilizing an electrostatic spinning process, and comprises the following steps:

1) weighing 2.0-2.5 g of Pluronic F-127(Pluronic F-127) material, adding into pre-cooled 8.0-10.0 g of complete culture medium, placing on a shaking table at 4 ℃ for 24 hours until the Pluronic F-127 material is fully dissolved, filtering the Pluronic F-127 solution by using a 0.22mm filter, placing on a refrigerator at 4 ℃ for 2 hours, spraying on a rotating device, and curing at room temperature for later use;

2) weighing 1.5-2.0 g of polyvinyl alcohol (PVA) material, slowly adding the PVA material into 15-20 g of deionized water under the condition of stirring, heating to about 80-100 ℃ after full swelling, accelerating dissolution, preserving heat for 3 hours to prepare uniform high molecular solution, sterilizing by high-temperature steam at 100 ℃, equally dividing into three parts, and filling one part into a second spray head for later use;

3) digesting cells of 3 rd generation New Zealand white rabbits with 0.25% trypsin, collecting the cells in a 50mL centrifuge tube, centrifuging for 5min at 1000r/min, discarding the supernatant, transferring the cell suspension to a sterilized PVA material, uniformly stirring to obtain a PVA solution containing cells, and filling the PVA solution into a first spray nozzle for later use;

4) digesting cells of a 4 th generation New Zealand white rabbit by using 0.25% trypsin, collecting the cells in a 50mL centrifuge tube, centrifuging for 5min at 1000r/min, removing supernatant, transferring cell suspension to a disinfected PVA material, uniformly stirring to obtain a PVA solution containing cells, and filling the PVA solution into a third nozzle for later use;

5) and 6-8kV direct-current voltage is loaded on the first spray head, the distance between the spray head and the rotating device is 6-8mm, and the negative electrode is loaded on the receiving platform. Continuously extruding the material at the stable flow rate of 150-180 mu L/min provided by a micro pump, rotating a rotating device at the speed of 800r/min, spinning for 1-3 hours, and wrapping the cured Pluronic F-127 material by the nano fiber to finish the preparation of the inner layer of the biological fiber ring;

6) and a direct-current voltage of 10-13kV is loaded on the second spray head, the distance between the spray head and the rotating device is 15-20mm, and the negative electrode is loaded on the receiving platform. Continuously extruding the material at a stable flow rate of 350-;

7) and 6-8kV direct-current voltage is loaded on the third nozzle, the distance between the third nozzle and the rotating device is 6-8mm, and the negative electrode is loaded on the receiving platform. Continuously extruding the material at the stable flow rate of 150-;

8) the rotary device is placed in a refrigerator at 4 ℃ for 1-2 hours, the Pluronic F-127 material is melted, and the biological fiber ring falling off from the rotary device is taken out.

The cells of the step 3) are chondrocytes or osteocytes.

The cells in the step 4) are muscle cells or fat stem cells.

Compared with the prior art, the invention has the following obvious and prominent substantive characteristics and remarkable advantages:

the biological fiber ring is in a concentric circle shape and has a three-layer structure; the fiber thickness of the middle layer is different from that of the inner layer and the outer layer, and the outer layer and the inner layer are respectively prepared from different cells and biodegradable materials; the whole structure of the biological fiber ring simulates a natural fiber ring, and the treatment time of the intervertebral disc protrusion is shortened. The method has the advantages of simple process, good controllability, high efficiency and the like.

Drawings

FIG. 1 is a schematic view of a bio-fiber ring preparation system.

FIG. 2 is a schematic view of the electrospinning process of a bio-fiber ring.

Fig. 3 is a schematic view of a structure of a bio-fiber ring.

Detailed Description

The preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings:

FIG. 1 is a schematic diagram of a system for preparing a bio-fiber ring. Wherein: the computer control system 1 is connected with the controller 2, the controller 2 is connected with the high-voltage power supply 6 and the micropump 3, the micropump 3 is connected with the spray head 4, the positive pole of the high-voltage power supply 6 is loaded on the needle head of the spray head 4, the negative pole of the high-voltage power supply 6 is loaded on the receiving platform 5, and the rotating device 7 is installed on the platform 5.

Example 1

The preparation method of the biological fiber ring comprises the following steps:

1) weighing 2.5 g of Pluronic F-127(Pluronic F-127) material, adding into 10.0 g of precooled complete culture medium, placing in a shaking table at 4 ℃ for 24 hours until the Pluronic F-127 material is fully dissolved, filtering the Pluronic F-127 solution by using a 0.22mm filter, placing in a refrigerator at 4 ℃ for 2 hours, spraying on a rotating device, and curing at room temperature for later use;

2) weighing 2.0 g of polyvinyl alcohol (PVA) material, slowly adding the PVA material into 20.0 g of deionized water under the condition of stirring, heating to about 100 ℃ after full swelling, accelerating dissolution, preserving heat for 3 hours to prepare uniform high molecular solution, sterilizing by high-temperature steam at 100 ℃, uniformly dividing into three parts, and filling one part into a second spray head for later use;

3) digesting cartilage cells of 3 rd generation New Zealand white rabbits with 0.25% trypsin, collecting the cartilage cells in a 50mL centrifuge tube, centrifuging for 5min at 1000r/min, discarding supernatant, transferring cell suspension to a sterilized PVA material, uniformly stirring to obtain a PVA solution containing cells, and filling the PVA solution into a first spray nozzle for later use;

4) digesting myocytes of a 4 th generation New Zealand white rabbit by using 0.25% trypsin, collecting the myocytes in a 50mL centrifuge tube, centrifuging for 5min at 1000r/min, removing supernatant, transferring cell suspension to a disinfected PVA material, uniformly stirring to obtain a PVA solution containing cells, and filling the PVA solution into a third nozzle for later use;

5) and 6kV direct-current voltage is loaded on the first spray head, the distance between the spray head and the rotating device is 6mm, and the negative electrode is loaded on the receiving platform. Continuously extruding the material at a constant flow rate of 150 mu L/min provided by a micro pump, rotating a rotating device at a speed of 800r/min, spinning for 1 hour, and wrapping the cured Pluronic F-127 material by the nano fiber to finish the preparation of the inner layer of the biological fiber ring;

6) and a 13kV direct-current voltage is loaded on the second spray head, the distance between the spray head and the rotating device is 20mm, and the negative electrode is loaded on the receiving platform. Continuously extruding the material at a stable flow rate of 380 μ L/min provided by a micro pump, rotating a rotating device at a speed of 1000r/min, and spinning for 1 hour to complete the preparation of the middle layer of the biological fiber ring;

7) and 6kV direct-current voltage is loaded on the third spray head, the distance between the spray head and the rotating device is 6mm, and the negative electrode is loaded on the receiving platform. Continuously extruding the material at a stable flow rate of 150 mu L/min provided by a micro pump, rotating a rotating device at a speed of 800r/min, and spinning for 1 hour to finish the preparation of the outer layer of the biological fiber ring;

8) the rotary device was placed in a refrigerator at 4 ℃ for 1 hour, the Pluronic F-127 material melted, and the bio-fiber rings that had fallen off the rotary device were removed.

Example 2

This example is essentially the same as example 1, except that: the cells adopted in the step 3) are bone cells, and the cells adopted in the step 4) are adipose-derived stem cells.