CN111910264A - Preparation method of bionic nerve nanofiber bundle - Google Patents

Abstract

The invention discloses a cell electrospinning preparation method of a bionic nerve nanofiber bundle. The method adopts the cell electrospinning technology to prepare the axially arranged cell-containing nanofiber bundle, and can realize integrated processing of degradable biological materials and cells. The method comprises the following specific steps: (1) preparing a uniform and degradable water-soluble high-molecular polymer solution by using deionized water; (2) sterilizing the polymer solution with high temperature steam; (3) adding the cell suspension into the sterilized polymer solution, and uniformly stirring; (4) and extracting solution containing cells by using a nozzle, arranging the nozzle on a micro pump, starting a high-voltage power supply and the micro pump, and performing electric spraying to prepare the axially-arranged nano fiber bundle containing the cells. The invention can accelerate the repair cycle of the living body, the whole preparation method is simple and easy to implement, and the invention has good application prospect in the field of peripheral nerve injury repair.

Description

Preparation method of bionic nerve nanofiber bundle

The technical field is as follows:

the invention discloses a cell electrospinning preparation method of a bionic nerve nanofiber bundle, and belongs to nanofiber bundle preparation technology in the field of tissue engineering and nerve repair engineering.

Background art:

the problem of repairing long-distance defects of peripheral nerves is a major technical obstacle faced by the present clinic, and the key of the problem lies in how to guide the axon growth of the nerves to correctly butt joint across the damaged section, namely, the axon of the motor nerves regenerated at the near end of the damage grows into the corresponding motor nerve bundle at the far end, and the axon of the sensory nerves regenerated at the near end grows into the corresponding sensory nerve bundle at the far end. If a bionic organism substitute can be used for guiding the nerve axon to grow directionally, the connection between the nerve axons at two ends becomes possible.

The cell electrospinning technology is characterized in that a biological material doped with living cells is used for preparing a cell-containing biological membrane, and the biological membrane has the advantages of uniform cell distribution, high porosity and good pore canal connectivity, and provides a good microenvironment for cell growth. Based on the technical characteristics and advantages, the invention adopts the technology to prepare the bionic biological substitute capable of guiding the directional growth of the nerve axon, the biological substitute is composed of axially arranged nano fiber bundles containing cells, the nano fiber bundles can guide the axial growth of the nerve axon and the growth of cells of defective tissues of the nano fiber bundles, and the mixed cells in the fiber bundles can effectively accelerate the repair progress of the tissues in the nerve defective area.

The invention content is as follows:

the invention adopts the following technical scheme:

the axially arranged nano fiber bundle containing cells is prepared by adopting a cell electrospinning technology, and the integrated processing of the degradable biological material and the cells can be realized. The method comprises the following steps:

(1) weighing 1.5-2.0 g of polyvinyl alcohol (PVA) by using an electronic balance, slowly adding the PVA into 15-20 g of deionized water under the condition of stirring, heating to about 80-100 ℃ after full swelling, accelerating dissolution, and preserving heat for 3 hours to prepare a uniform high molecular solution;

(2) sterilizing the above materials with high temperature steam at 120 deg.C;

(3) digesting the rat neural stem cells of the 4 th generation 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 the sterilized PVA material, uniformly stirring to obtain PVA solution containing cells, and filling the PVA solution into a spray head for later use;

(4) the spray head is loaded with 6-8KV DC voltage, the distance between the spray head and the rotary receiving device is 6-8mm, and the negative pole is loaded on the backing plate. The material is provided by a micro pump at the stable flow rate of 150-;

(5) and (4) removing the nano fibers to bundle two ends into fiber bundles which are arranged in parallel.

The cells in the step (3) are Schwann cells or neuron cells.

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

(1) the invention prepares the axially arranged nano fiber bundle containing cells, the cells are uniformly distributed, and the signal transmission between the cells and the fiber bundle is facilitated.

(2) The invention prepares the axially arranged nano fiber bundle containing cells, which is beneficial to guiding the directional growth of the nerve axon.

(3) The method has the advantages of simple process, good controllability, high efficiency and the like.

Description of the drawings:

FIG. 1 is a schematic diagram of a system for preparing a biomimetic nerve nanofiber bundle.

FIG. 2 is a schematic diagram of a cell electrospinning process.

Wherein: the 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.

The specific implementation mode is as follows:

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

the first embodiment is as follows:

(1) weighing 1.76 g of PVA particles by using an electronic balance, slowly adding the PVA particles into 18.24 g of deionized water under the condition of stirring, heating to about 90 ℃ after full swelling, accelerating dissolution, and preserving heat for 3 hours to prepare a transparent 8.8% PVA solution;

(2) sterilizing the above materials at 120 deg.C;

(3) digesting the rat neural stem cells of the 4 th generation 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 the sterilized materials, and uniformly stirring to obtain PVA solution containing cells;

(4) connecting a computer control system, a controller, a high-voltage power supply, a base plate, a rotating device, a spray head and a micro pump as shown in figure 1;

(5) 8mL of PVA solution containing cells is extracted by a 10mL nozzle, and the nozzle is arranged on a micro pump;

(6) starting a high-voltage power supply, loading 6.5KV of direct-current voltage, adjusting the distance between the needle head and the receiving platform to be 20cm,

(7) the feeding speed is 160ul/min, and the nano-fiber is prepared by spinning for 2 hours;

(8) and after spinning is finished, taking down two ends of the nanofiber, bundling the nanofiber into a bundle, putting the nanofiber into a culture dish, adding a culture solution, and putting the nanofiber into an incubator for culture. Finally obtaining the axially arranged nano fiber bundle containing cells.

Example two:

this example is substantially the same as example one, except that: weighing 2 g of PEO particles by using an electronic balance, slowly adding the PEO particles into 8 g of deionized water under the condition of stirring, heating to about 60 ℃ after the PEO particles are fully swelled, accelerating dissolution, and preserving heat for 3 hours to prepare a transparent 20% PEO solution;

example three:

this example is substantially the same as example one, except that: the cells adopted in the step (4) are Schwann cells.

Claims (2)

1. The preparation method of the bionic nerve nanofiber bundle comprises the following steps of preparing the bionic nerve nanofiber bundle by using a cell electrospinning process, wherein the fiber bundle consists of degradable high-molecular polymers and cells:

(1) weighing 1.76 g of PVA particles by using an electronic balance, slowly adding the PVA particles into 18.24 g of deionized water under the condition of stirring, heating to about 90 ℃ after full swelling, accelerating dissolution, and preserving heat for 3 hours to prepare a transparent 8.8% PVA solution;

(2) sterilizing the above materials at 120 deg.C;

(3) digesting the rat neural stem cells of the 4 th generation 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 the sterilized materials, and uniformly stirring to obtain PVA solution containing cells;

(4) connecting a computer control system, a controller, a high-voltage power supply, a base plate, a rotating device, a spray head and a micro pump as shown in figure 1;

(5) 8mL of PVA solution containing cells is extracted by a 10mL nozzle, and the nozzle is arranged on a micro pump;

(6) starting a high-voltage power supply, loading 6.5KV of direct-current voltage, adjusting the distance between the needle head and the receiving platform to be 20cm,

(7) the feeding speed is 160ul/min, and the nano-fiber is prepared by spinning for 2 hours;

(8) and after spinning is finished, taking down two ends of the nanofiber, bundling the nanofiber into a bundle, putting the nanofiber into a culture dish, adding a culture solution, and putting the nanofiber into an incubator for culture. Finally obtaining the axially arranged nano fiber bundle containing cells.

2. The method for producing a fiber bundle according to claim 1, characterized in that: the cells in the step 3) are Schwann cells or neuron cells.

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