CN109224133B - Preparation method of biological multilayer nerve conduit containing mesenchymal stem cells - Google Patents

Preparation method of biological multilayer nerve conduit containing mesenchymal stem cells Download PDF

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CN109224133B
CN109224133B CN201811107450.5A CN201811107450A CN109224133B CN 109224133 B CN109224133 B CN 109224133B CN 201811107450 A CN201811107450 A CN 201811107450A CN 109224133 B CN109224133 B CN 109224133B
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CN109224133A (en
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尹俊
俞梦飞
刘婧懿
严梦玲
傅建中
吴俊杰
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Zhejiang University ZJU
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Abstract

The invention discloses a preparation method of a biological multilayer nerve conduit containing mesenchymal stem cells, which comprises the following steps: (1) dissolving the photo-curable biological hydrogel in PBS solution containing a photoinitiator to prepare biological ink with two concentrations; (2) collecting mesenchymal stem cells with the cell confluency of more than 80%, and after treatment, using low-concentration biological ink to resuspend the mesenchymal stem cells to obtain cell-loaded biological ink; (3) respectively loading cell-loaded biological ink and high-concentration biological ink into two nozzles of a biological 3D printing device; (4) setting printing parameters, and firstly printing cell-carrying biological ink on an optical axis rotating at a constant speed to obtain a first layer of nerve conduit; then printing high-concentration biological ink to obtain a double-layer hollow tubular nerve conduit; (5) and irradiating ultraviolet light to obtain the biological multilayer nerve conduit. The invention has the advantages of short and simple preparation time, mild operation conditions, simple structure of the prepared nerve conduit, good biocompatibility and degradability.

Description

Preparation method of biological multilayer nerve conduit containing mesenchymal stem cells
Technical Field
The invention belongs to the field of design and preparation of nerve conduits, and particularly relates to a preparation method of a biological multilayer nerve conduit containing mesenchymal stem cells.
Background
The nervous system (nervous system) is a system that plays a leading role in regulating physiological functional activities in the body, and is mainly composed of nervous tissues, and is divided into two major parts, the central nervous system and the peripheral nervous system. Various traumas such as compression, stretching, laceration and the like and other factors such as tumor, ischemia and the like are easy to cause damage to the nervous system, thereby causing the functional deficiency of the nervous system and other neurological diseases, and seriously reducing the quality of work and life of patients. Currently, the mainstream nerve repair techniques include the following: direct anastomosis, autologous or allogeneic nerve transplantation, and repair via nerve conduits. The direct anastomosis can only be used for the condition that the neurotmesis is short (<8mm), and the curative effect is not high in good rate; the autologous or allogeneic nerve transplantation can cause the loss of the function of the nerve in the supply area, and has the problems of secondary operation and the like, while the nerve conduit repair (bridging) can make up the defects of the two technologies and attract the attention of more and more researchers and doctors.
Chinese patent publication No. CN108379667A discloses a novel biodegradable nerve conduit and a preparation technique thereof, and proposes a method for preparing a tubular nerve conduit from one or more of poly (lactic-co-glycolic acid), poly (ethylene carbonate), poly (lactic acid) and poly (glycolic acid). The biological material adopted by the preparation method is degradable, the embedding pressure on nerve growth cannot be caused, and the preparation method is simple and easy to operate. However, the method adopts a solution replacement method which is time-consuming for preparing the nerve conduit, only needs 1.5-2h and 36-48h in the heating and drying processes respectively, and cannot meet the urgent nerve conduit requirement; and the manufacturing process adopted can only prepare the nerve conduit with a simple structure, and the microstructure of the nerve conduit, such as the pore size, the pore separation and the porosity of the nerve conduit, cannot be designed.
With the continuous development of tissue engineering, researchers find that nerve growth factors or seed cells are beneficial to accelerating the shape and functional recovery of peripheral nerves, and then the preparation of nerve conduits containing the nerve growth factors or the seed cells is gradually promoted. Chinese patent publication No. CN105597148A discloses a method of a nerve scaffold for nerve injury repair. The nerve scaffold comprises a collagen scaffold, nerve stem cells cultured on the collagen scaffold, and growth factors fixed on the collagen scaffold by heparin. The specific preparation process of the method is as follows: firstly, preparing a collagen solution; then pouring the solution into a grinding tool, and performing freeze-drying sterilization to obtain collagen sponge; sequentially soaking the collagen sponge in a cross-linking solution containing heparin and a growth factor solution; and finally, primarily culturing the neural stem cells on the neural bracket to obtain the neural catheter carrying the cells and the growth factors. The preparation method has the advantages of easily-accessible raw materials and simple steps, avoids acid-base neutralization and dialysis, reduces the probability of contacting collagen material with harmful reagent, and improves the safety of the obtained nerve scaffold. However, the attachment position and distribution density of the growth factors and the neural stem cells on the nerve conduit are random and uncontrollable, and the nerve conduit with the uniform distribution, gradient distribution or programmed distribution of the neural stem cells and the neural growth factors cannot be obtained.
Disclosure of Invention
The invention aims to provide a preparation method of a biological multilayer nerve conduit containing mesenchymal stem cells, which has the advantages of short and simple preparation time consumption, mild operation conditions, simple structure of the prepared nerve conduit, good biocompatibility, degradability and no embedment pressure on nerve growth.
A preparation method of a biological multilayer nerve conduit containing mesenchymal stem cells comprises the following steps:
(1) dissolving the photo-curable hydrogel in PBS (phosphate buffer solution) containing a photoinitiator to prepare two types of bio-ink with low concentration and high concentration;
(2) collecting mesenchymal stem cells with the cell confluency of more than 80% in a culture dish, digesting and centrifuging, and then using the low-concentration biological ink prepared in the step (1) to resuspend the mesenchymal stem cells to obtain cell-loaded biological ink;
(3) respectively loading the cell-loaded biological ink and the high-concentration biological ink prepared in the step (1) into two nozzles of a biological 3D printing device, and enabling the biological ink to be gelatinous through post-treatment;
(4) setting printing parameters of a biological 3D printing device, and firstly printing cell-carrying biological ink on an optical axis rotating at a constant speed according to a preset instruction to obtain a first layer of nerve conduit; then printing high-concentration biological ink on the surface of the first layer of nerve conduit to obtain an initial double-layer hollow tubular nerve conduit;
(5) and carrying out ultraviolet irradiation on the obtained initial double-layer hollow tubular nerve conduit to obtain the biological type multilayer nerve conduit.
The nerve conduit with stable structure and good mechanical strength is prepared by cooling crosslinking, a biological 3D printing technology based on extrusion molding and ultraviolet curing, the whole manufacturing process is short in time consumption, simple and convenient to operate, and mild in working conditions. The nerve conduit prepared by the method can be immediately transferred into the body to help treat the nerve defect, and can also be transferred into the body after being cultured for a certain time in vitro.
Preferably, the photocurable biohydrogel used in step (1) is methacrylic gelatin. The hydrogel is prepared into sterile methacrylic acid gelatin solid through filtration sterilization and freeze drying.
The methacrylic acid gelatin is a biodegradable synthetic polymer, the degradation product is harmless, and the degradation rate of the prepared nerve conduit can be controlled by controlling the concentration of the biological ink prepared from the methacrylic acid gelatin. Meanwhile, the methacrylic acid gelatin has temperature sensitivity and can be crosslinked in a physical mode of reducing the temperature of the ink.
Preferably, the photoinitiator used in step (1) is one of conventional 2-hydroxy-4- (2-hydroxyethoxy) -2-methylpropanedione (Irgacure 2959) or lithium phenyl-1, 4-6-trimethylbenzoylphosphonate (LAP).
Further, the concentration of the photoinitiator in the PBS solution is 0.05% to 0.1% (w/v).
Preferably, in the step (1), the concentration of the methacrylic acid gelatin in the low-concentration bio-ink is 5% to 15% (w/v), and the concentration of the methacrylic acid gelatin in the high-concentration bio-ink is 20% to 30% (w/v). Further preferably, the concentration of methacrylic gelatin in the low-concentration bio ink is 5% (w/v), and the concentration of methacrylic gelatin in the high-concentration bio ink is 30% (w/v).
Preferably, in step (2), the mesenchymal stem cell is boneMesenchymal Stem Cells (BMSCs). The mesenchymal stem cell density in the cell-carrying biological ink is 1.0 x 106-1.0*107ml-1。
Preferably, in the step (3), the temperatures of the two nozzles of the biological 3D printing device are respectively 1-3 ℃ lower than the gel point temperatures of the cell-carrying biological ink and the high-concentration biological ink. The temperature of the biological ink in the spray head is indirectly controlled by controlling the temperature of the equipment for placing the spray head, and the temperature of the ink is reduced to be in a gel state through the low-temperature crosslinking characteristic of the methacrylic acid gelatin, so that the silk is conveniently extruded.
In the step (4), during printing, the filament extruded from the nozzle is adhered to an optical axis which rotates at a certain angular speed and is tightly connected with the motor, and the nozzle does reciprocating motion in the direction of the axis of the optical axis of the motor.
Preferably, the rotation speed of the optical axis is 15-25rpm, and the moving speed of the nozzle is 100-300 mm/min. Further preferably, the rotation speed of the optical axis is 20rpm, and the movement speed of the head is 200 mm/min.
The outer diameter of the optical axis is 1-5mm, and the inner diameter of the needle head of the spray head is 159 and 603 mu m.
Preferably, in the step (5), the wavelength of the ultraviolet light is 365nm, and the irradiation density of the ultraviolet light is 2W/cm2And the illumination time is 30-60 s.
The mechanical strength of the inner layer of the finally prepared nerve conduit is lower than that of the outer layer. The inner layer has low mechanical strength, is beneficial to attaching, spreading, propagating and differentiating the mesenchymal stem cells and promotes the repair of the nerve defects; the outer layer has high mechanical strength, plays a supporting role and maintains the stability of the whole catheter.
The compression modulus of the inner layer of the nerve conduit is 2-200KPa, and the tensile modulus is 2-40 KPa; the outer layer has a mechanical compression modulus of 350-700KPa and a tensile modulus of 50-150 KPa.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention adopts the material, namely methacrylic acid gelatin (GelMA), which is a product obtained by grafting reaction of gelatin, has excellent biocompatibility, biodegradability, temperature sensitivity and the like of the gelatin, and also has the characteristic of photocuring. Therefore, the nerve conduit is cured by ultraviolet light, has very mild operation conditions, does not threaten the physical health of operators, does not contact a plurality of toxic reagents, and enhances the safety of the conduit.
2. The degradation performance of the material adopted in the invention, namely the methacrylic acid gelatin (GelMA), can be indirectly adjusted by adjusting the concentration of the biological ink, so that the requirements of the medical nerve conduits with different degradation times can be completely met.
3. The invention integrally adopts the biological 3D printing technology based on extrusion molding to prepare the initial double-layer nerve conduit and then carries out ultraviolet curing, the time consumption of the whole preparation process is quite short, and the urgent nerve conduit requirement is completely met. And through the 3D printing technology, the structure of the nerve conduit can be artificially controlled, such as porosity, pore size, conduit length, inner and outer layer thickness and the like, and the distribution of mesenchymal stem cells in the inner layer of the conduit. Simultaneously, according to the 3D printing system, the structure and the shape of the catheter can be designed according to the condition of a patient.
Drawings
FIG. 1 is a schematic flow diagram of the present invention;
FIG. 2 is a schematic structural diagram of a biological 3D printing device required by the preparation method of the invention;
FIG. 3 is an enlarged view of a portion of area A of FIG. 2;
FIG. 4 is a schematic structural view of a biotype double-layer nerve conduit manufactured by the present invention.
Detailed Description
The present invention is further illustrated by the following specific examples and the accompanying drawings, and the present invention is not limited to the examples, and the non-essential modifications and adjustments of the neural stent by the researchers based on the above descriptions still belong to the protection scope of the present invention.
As shown in fig. 2-3, it is a schematic structural diagram of a biological 3D printing apparatus required by the preparation method of the present invention. The whole device comprises: print shower nozzle 1, print shower nozzle 2 No. two, motor 3, optical axis 4, ultraviolet irradiation equipment 5, light pipe 6. During printing, the motor 3 drives the optical axis 4 to rotate at a set rotating speed, the first printing nozzle 1 moves back and forth along the axis direction of the optical axis 4, the extruded gel GelMA wire 7 is adhered to the rotating optical axis 4, and a first layer of nerve conduit is printed; subsequently, the second print head 2 prints a second layer of nerve conduits along the path of the first print head. After printing the two layers of nerve conduits, the ultraviolet illumination device 5 is opened, and ultraviolet illumination is carried out on the nerve conduits through the light conduit 6 to obtain the required multilayer nerve conduits.
Example 1
As shown in fig. 1, a method for preparing a biological multilayer nerve conduit containing mesenchymal stem cells comprises the following specific steps:
(1) a defined mass of sterile GelMA was weighed into a 10ml centrifuge tube and dissolved using 0.1% (w/v) LAP in PBS to prepare 5% (w/v) and 30% (w/v) GelMA bio-inks in that order.
(2) Collecting bone marrow mesenchymal stem cells (BMSC) with the cell confluency of 80% or more in the culture dish, digesting the cells by using 0.25% (w/v) pancreatin solution, then carrying out centrifugation treatment, and after the digestion and the centrifugation treatment, carrying out centrifugation at the speed of 100rpm for 5 min. Pouring out supernatant in the centrifuge tube, using 5% (w/v) GelMA bio-ink prepared in the step (1) to re-suspend cells, gently blowing and beating the cells to uniformly distribute the cells in the bio-ink, wherein the cell density in the bio-ink is 1.0 x 106And obtaining the 5% (w/v) GelMA bio-ink carrying the cells.
(3) Introducing 5% (w/v) GelMA biological ink carrying cells into a first printing nozzle 1 and placing the first printing nozzle on a printer; setting the temperature of the equipment for placing the nozzle at 10 deg.C, and printing after 10 min.
Filling 30% (w/v) GelMA biological ink prepared in the step (1) into a second printing nozzle 2, and putting the second printing nozzle into a printer; setting the temperature of the equipment for placing the spray head at 26 ℃, and printing after 10min when the biological ink in the spray head is in gel state.
(4) The moving speed of the spray heads is set to be 200mm/min, the two spray heads uniformly select a 26G needle head (the inner diameter is 0.260mm), the rotating speed of the motor is 20rpm, and the diameter of the optical axis of the motor is 3 mm. After positioning, the cell-loaded 5% (w/v) GelMA bio-ink was first printed. And (3) printing 30% (w/v) GelMA biological ink after the 5% (w/v) GelMA biological ink spray head moves 50mm along the axial direction of the optical axis tightly connected with the motor. Similarly, the 30% (w/v) GelMA bio-ink jet motion repeated 5% (w/v) GelMA bio-ink jet path and ended printing, resulting in an initial double-layer hollow nerve conduit 50mm long, with an inner layer diameter of 3mm, an outer layer diameter of 4.040mm, an inner layer thickness of 0.260mm, and an outer layer thickness of 0.260mm, as shown in fig. 4.
(5) Carrying out ultraviolet curing on the nerve conduit obtained in the step (4), wherein the wavelength of ultraviolet light is 365nm, and the ultraviolet illumination density is 2W/cm2The violet light irradiation time was 30 s. After illumination, the nerve conduit GelMA is subjected to irreversible crosslinking, and the structure is stable.
Under the same condition, 6 double-layer nerve conduits are prepared for testing, the mean value of the compression modulus of the inner layer is 2.86KPa, and the mean value of the tensile modulus is 2.08 KPa; the average value of the mechanical compression modulus of the outer layer is 700KPa, and the average value of the tensile modulus is 150 KPa.
The cells are lightly taken off from the motor light, can be immediately transplanted into the body to repair the nerve defects, and can also be transplanted into the body after being cultured in vitro for a period of time.
Compared with the nerve conduit which does not contain cells and has the same size and structural design, the nerve conduit with the BMSC in the inner layer prepared by the preparation method is soaked in the cell suspension of the nerve cells, the number and the cell morphology of the nerve cells attached to the inner surface of the nerve conduit after a period of time, and the consistent rate of the cell growth direction is obviously superior to that of a control group.
Example 2
The difference from example 1 is that in step (1), the concentration of the low-concentration GelMA bio-ink was 15%, and the other conditions were not changed. 6 double-layer nerve conduits are prepared and tested, the mean value of the compressive modulus of the inner layer of the obtained nerve conduit is 250KPa, and the mean value of the tensile modulus is 37.5 KPa; the average value of the mechanical compression modulus of the outer layer is 700KPa, and the average value of the tensile modulus is 150 KPa.
Example 3
The difference from example 1 is that, in step (4), a 22G needle (inner diameter 0.413mm) is uniformly selected for two nozzles, and other conditions are not changed, so that an initial double-layer hollow nerve conduit with the length of 50mm, the inner layer diameter of 3mm, the outer layer diameter of 4.652mm, the inner layer thickness of 0.413mm and the outer layer thickness of 0.413mm is obtained.
And (3) carrying out ultraviolet curing to obtain a final double-layer nerve conduit, and testing the mechanical compression modulus and the tensile modulus of the inner layer material and the outer layer material of the obtained double-layer nerve conduit, wherein the test results are basically unchanged compared with the test results in the embodiment 1.
Example 4
The difference from example 1 is that, in step (4), 5mm of the diameter of the optical axis of the motor was used, and other conditions were not changed, to obtain an initial double-layered hollow nerve conduit having a length of 50mm, an inner layer diameter of 5mm, an outer layer diameter of 6.040mm, an inner layer thickness of 0.260mm, and an outer layer thickness of 0.260 mm.
And (3) carrying out ultraviolet curing to obtain a final double-layer nerve conduit, and testing the mechanical compression modulus and the tensile modulus of the inner layer material and the outer layer material of the obtained double-layer nerve conduit, wherein the test results are basically unchanged compared with the test results in the embodiment 1.
Example 5
The difference from the embodiment 1 is that in the step (4), 30% (w/v) GelMA bio-ink is printed after the 5% (w/v) GelMA bio-ink nozzle moves 100mm along the axial direction of the optical axis tightly connected with the motor. Similarly, the 30% (w/v) GelMA bio-ink jet movement repeated 5% (w/v) GelMA bio-ink jet path and ended printing, resulting in an initial double-layer hollow nerve conduit 100mm long, with an inner layer diameter of 3mm, an outer layer diameter of 4.040mm, an inner layer thickness of 0.260mm, and an outer layer thickness of 0.260 mm.
And (3) carrying out ultraviolet curing to obtain a final double-layer nerve conduit, and testing the mechanical compression modulus and the tensile modulus of the inner layer material and the outer layer material of the obtained double-layer nerve conduit, wherein the test results are basically unchanged compared with the test results in the embodiment 1.

Claims (8)

1. A preparation method of a biological multilayer nerve conduit containing mesenchymal stem cells is characterized by comprising the following steps:
(1) dissolving the photo-curable biological hydrogel in PBS solution containing a photoinitiator to prepare two types of biological ink with low concentration and high concentration; the light-curable biological hydrogel is methacrylic acid gelatin, the concentration of the methacrylic acid gelatin in the low-concentration biological ink is 5-15% (w/v), and the concentration of the methacrylic acid gelatin in the high-concentration biological ink is 20-30% (w/v);
(2) collecting mesenchymal stem cells with the cell confluency of more than 80% in a culture dish, digesting and centrifuging, and then using the low-concentration biological ink prepared in the step (1) to resuspend the mesenchymal stem cells to obtain cell-loaded biological ink;
(3) respectively loading the cell-loaded biological ink and the high-concentration biological ink prepared in the step (1) into two nozzles of a biological 3D printing device to enable the biological ink to be gelatinous;
(4) setting printing parameters of a biological 3D printing device, and firstly printing cell-carrying biological ink on an optical axis rotating at a constant speed according to a preset instruction to obtain a first layer of nerve conduit; then printing high-concentration biological ink on the surface of the first layer of nerve conduit to obtain an initial double-layer hollow tubular nerve conduit;
(5) and carrying out ultraviolet irradiation on the obtained initial double-layer hollow tubular nerve conduit to obtain the biological type multilayer nerve conduit.
2. The method for preparing a biologic multilayer nerve conduit containing mesenchymal stem cells according to claim 1, wherein in the step (1), the photoinitiator is one of 2-hydroxy-4- (2-hydroxyethoxy) -2-methylpropanedione and lithium phenyl-1, 4-6-trimethylbenzoylphosphonate, and the concentration of the photoinitiator in the PBS solution is 0.05-0.1% (w/v).
3. The method for preparing a biologic multilayer nerve conduit containing mesenchymal stem cells according to claim 1, wherein in step (2), the mesenchymal stem cells are bone marrow mesenchymal stem cells.
4. The method for preparing a biologic multilayer nerve conduit containing mesenchymal stem cells according to claim 1, wherein in step (2), the density of the mesenchymal stem cells in the cell-loaded biologic ink is 1.0 × 106-1.0×107ml-1。
5. The method for preparing a biological multilayer nerve conduit containing mesenchymal stem cells according to claim 1, wherein in the step (3), the temperature of two nozzles of the biological 3D printing device is 1-3 ℃ lower than the gel point temperature of the cell-carrying biological ink and the high-concentration biological ink respectively.
6. The method for preparing a biologic multilayer nerve conduit containing mesenchymal stem cells according to claim 1, wherein in the step (4), the nozzle reciprocates along the axial direction of the optical axis during the printing process, the rotation speed of the optical axis is 15-25rpm, and the moving speed of the nozzle is 100-300 mm/min.
7. The method for preparing a biologic multilayer nerve conduit containing mesenchymal stem cells according to claim 6, wherein the outer diameter of the optical axis is 1-5mm, and the inner diameter of the needle of the nozzle is 159-603 μm.
8. The method for preparing a biologic multilayer nerve conduit containing mesenchymal stem cells according to claim 1, wherein in the step (5), the wavelength of the ultraviolet light is 365nm, and the irradiation density of the ultraviolet light is 2W/cm2And the illumination time is 30-60 s.
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